main steps in dna double-strand break repair: an introduction to...
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Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes Postprint (Author accepted manuscript)
Ranjha, Lepakshi; Institute for Research in Biomedicine (IRB), Faculty of Biomedical Sciences, Università della Svizzera italiana, Switzerland
Howard, Sean Michael; Institute for Research in Biomedicine (IRB), Faculty of Biomedical Sciences, Università della Svizzera italiana, Switzerland
Cejka, Petr; Institute for Research in Biomedicine (IRB), Faculty of Biomedical Sciences, Università della Svizzera italiana, Switzerland - Institute of Biochemistry, Department of Biology, ETH Zürich, Switzerland
Originally published in:
Chromosoma
2018 / Vol. 127 / n. 2 (June) / pp. 187–214
Published version: https://doi.org/10.1007/s00412-017-0658-1
Online publication: 2018-01-11
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Manuscripttitle:
Main steps in DNA double-strand break repair: an introduction to homologous
recombinationandrelatedprocesses
Authors:
LepakshiRanjha1*,SeanHoward1*,PetrCejka1,2
Affiliation:
1Institute for Research in Biomedicine Università della Svizzera Italiana, Bellinzona,
Switzerland
2DepartmentofBiology,InstituteofBiochemistryETHZurich,Zurich,Switzerland
Correspondence:
PetrCejka,[email protected],phone:+41918200342,fax:+41918200305
*Equalcontribution
Keywords:Homologousrecombination,end-joining,DNAdouble-strandbreakrepair,
meiosis,replicationstress,DNAendresection,DNAstrandexchange
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Abstract
DNA double-strand breaks arise accidentally upon exposure of DNA to radiation,
chemicals or result from faulty DNA metabolic processes. DNA breaks can also be
introduced in a programmedmanner, such as during thematuration of the immune
system,meiosis or cancer chemo- or radiotherapy. Cells have developed a variety of
repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly,
vegetativecellsemploymechanismsthatrestorethe integrityofbrokenDNAwith the
highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or
developing lymphocytes exploitDNAbreakage to generatediversity.Here,we review
the main pathways of eukaryotic DNA double-strand break repair with the focus on
homologousrecombinationanditsvarioussub-pathways.Wehighlightthedifferences
between homologous recombination and end-joining mechanisms including non-
homologous end-joining andmicrohomology-mediated end-joining, and offer insights
intohowthesepathwaysareregulated.Finally,weintroducenon-canonicalfunctionsof
therecombinationproteins,inparticularduringDNAreplicationstress.
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1OverviewofDNAdouble-strandbreakrepairpathways
1.1FormationandtypesofDNAbreaksOurDNA is under a constant threat of damage from radiation, chemicals or aberrant
DNAmetabolicprocesses(JacksonandBartek2009;CicciaandElledge2010;Tubbsand
Nussenzweig2017).AlargefractionofDNAlesionsinvolvemodificationsofDNAbases,
suchasoxidation,ultravioletlight-inducedpyrimidinedimers,methylationorcreation
of abasic sites, which leave the phosphodiester backbone intact. Other abnormalities
resultinthedisruptionofthephosphodiesterbackbone.Themostcommonoftheseare
DNA single-strandbreaks(SSBs),whereonlyoneDNA strand is interrupted (Fig.1a).
SSBs normally do not compromise the integrity of double-stranded DNA (dsDNA).
However, ifanSSBisleftunrepairedandthelesionisencounteredbyDNAmachinery
that separates the DNA duplex into two component single-strands (ssDNA), such as
duringDNAreplication,anSSBcanbeconvertedintoaone-endedDNAdouble-strand
break(DSB),seeFig.1b.BothSSBsandDSBscanariseasaresultofionizingradiation
(IR), which may occur either directly or indirectly via generation of reactive oxygen
species (Ward 1988). As a result, radiation-induced DNA damage results in complex
lesions,wherebothSSBsandDSBsareaccompaniedbyoxidativeDNAdamage(Oliveet
al. 1990; Olive 1998). The most common source of accidental IR exposure is the
radioactive radon gas that accumulates in certain locations in the basements of old
homes (Jackson and Bartek 2009). IR remains one of the most effective treatments
during anti-cancer therapy, as it preferentially affects rapidly dividing cancer cells
(JacksonandBartek2009;Baskaretal.2014).SSBsandDSBsalsoariseduringaberrant
DNA topoisomerase reactions, which can occur spontaneously or upon exposure to
specific inhibitors that are often used as anti-cancer chemotherapeutics (Holm et al.
1989;Canela et al. 2017). Finally,DNAbreaksunder certain conditions result froma
nucleaseattackingdiverseDNA intermediates, includingstalledDNAreplication forks.
Aswillbe introduced inchapter7,DSB-likestructuremayalsoariseduringaprocess
termedreplicationforkreversalwithouttemplateDNAbreakage.
Dependingonthemechanismofbreakformation,aDSBcaneitherbeone-endedor
two-ended(Fig.1b,c).Mostone-endedDSBsarisewhenDNAreplicationencountersan
SSBandthereplicationforkfallsapart,orwhenaDNAreplicationforkstallsandoneof
thearmsiscleavedbyanuclease.Two-endedDSBstypicallyformwhenbothstrandsof
linear dsDNA are broken simultaneously, or when two ssDNA breaks form in an
immediate proximity; in this case, the broken end would contain a short stretch of
overhangingssDNA.Additionally, dependingon themechanismof theDSB formation,
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DNAendsmaybeeitherchemically"clean"or"dirty".Theso-calledcleanDNAbreaks,
apart from the broken phosphodiester backbone, bear normal DNA chemistry. Dirty
DNA ends instead contain additional adducts that may include anything from small
chemicalgroupstocovalentlyattachedproteins.DNAbreaksinducedbyDNAnucleases
aregenerallyclean,butmanyDSBsinducedbyionizingradiationaredirty(Oliveetal.
1990). Likewise, DSBs induced by aberrant DNA topoisomerase reactions result in a
covalently attachedDNA topoisomeraseat thebreak end(Tse et al. 1980).Aswill be
describedbelow,themechanismofDSBrepairdependslargelyonwhetherthebreakis
one-ortwo-ended,chemicallycleanordirty,aswellasthecellcyclestage.
OurcellshavedevelopednumerousmechanismsforrepairingDSBswithaminimal
loss of genetic information. However, if a DSB is repaired incorrectly, it can lead to
mutationsandchromosomalrearrangements,resultinginaberrantregulationofcellular
growth and cancer development or even cell death (Jackson and Bartek 2009).
Therefore, accurate recognition and repair of DSBs is essential to maintain genomic
integrityandpreventtumorigenesis.
1.2End-joiningandhomologousrecombinationmechanismsrepairDNAdouble-
strandbreaks
Eukaryotic cells use twomainprocesses for DSB repair, end-joining and homologous
recombination(HR),seeFig.2a.Theend-joiningpathwayscanbe furtherdivided into
canonical non-homologous end-joining (NHEJ) and alternative non-homologous end-
joining(altNHEJ),alsotermedmicrohomology-mediatedend-joining(MMEJ).TheMMEJ
abbreviation will be used hereafter in this review. As the name indicates, NHEJ and
MMEJ involve thedirectligationoftwoDSBendswithlittleornosequencehomology
required, see below for details (Chang et al. 2017). Therefore, a key feature of end-
joiningisthatarepairtemplate,suchasthesisterchromatid, isnotrequired,soitcan
occurduringanyphaseofthecellcycle.BothNHEJandMMEJprocessestypicallyleadto
a limited loss of genetic information resulting in short deletions at the DSB site.
Additionally, becauseNHEJandMMEJmechanismsare template-independent, ligation
of the incorrect ends, if multiple DSBs are present, can generate large deletions or
chromosomalrearrangements(Changetal.2017).The end-joiningpathwayscanonly
repair two-ended DSBs, and abnormal structures at the break sites ("dirty" ends,
especially protein blocks) may inhibit this type of repair. In summary, end-joining
pathwaysrepresentfast,butpotentiallymutagenicDSBrepairprocesses(Fig.2a).
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In contrast, HR requires a homologous sequence as a template for repair
(Kowalczykowski 2015). This allows the recombination machinery to restore any
missinggeneticinformationinthevicinityofthebreaksite,and,asaresult,HRislargely
accurate.Inmostcasesinvegetativelygrowingcells,thesisterchromatidisusedasthe
repair template. This restricts recombination to cell cycle stages when the sister
chromatidisavailable,whichincludestheSandG2phases,andthusnecessitatesastrict
controlmechanism(Orthweinetal.2015).HRiscapableofrepairingbothone-andtwo-
endedDSBs and canalso repairdirtyDNAbreaks; inparticular thosewith covalently
attachedproteins. In contrast to end-joining,HR ismechanisticallymore complicated,
involves a larger number of enzymes, and is thus comparatively slower but more
accurate(Kowalczykowski2015;Changetal.2017).
Recent yearsbroughtbreakthroughs in genomeediting technologies,whichwere
spearheadedbythedevelopmentofengineerednucleasessuchaszincfingernucleases
(ZFNs) or transcription-activator like effector nucleases (TALENs) (Lombardo et al.
2007;Bedelletal.2012).Themajorityofgenomeeditingapplicationsnowexploitthe
bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9
system (Jinek et al. 2012; Mali et al. 2013). The common denominator of these
approaches is the capacity to induce a site-specificDSB.The choiceof theDSB repair
pathwaythendictatestheresultofediting(Fig.2b).ImpreciserepairbyNHEJorMMEJ
givesriseto"indel"mutations(insertionordeletions,althoughdeletionsaremuchmore
common)at thebreaksite,whichmaydisrupt thereading frameof the targetedgene
and thus result in a loss of function. Conversely, if a DNA template is provided, the
recombinationmachinerymaygetinvolved,whichcanmediateprecisealterationofthe
DNA sequence, including introductionofDNA segmentsor correctionof apathogenic
mutation(Fig.2b).Theadvanceofthesegenomeeditingtechnologiesbroughtrenewed
interest in understanding the balance between the DSB repair pathways, as the
inhibitionofMMEJandNHEJrepairpromotesHR-basedprecisegenomeediting(Chuet
al.2015;Mateos-Gomezetal.2017;Schimmeletal.2017;Zelenskyetal.2017).
Thekeyprocess thatstandsat thecrossroadsbetweenend-joiningandHR is the
initialprocessingoftheDNAbreak(Cejka2015).NHEJandMMEJrequirelittleDNAend
processing (see chapter 2 for more details). In contrast HR (including all its sub-
pathways) is initiated by DNA resection at the break site that exposes long tracts of
ssDNA.ThisssDNAisthenusedinasearchforahomologousdsDNAsequence(suchas
thesisterchromatid)thatservesasatemplateforthelargelyaccuraterepairoftheDSB
by the recombinationpathway.At the same time, extendedDNAend resectionmakes
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the DNA break generally non-ligatable and inhibits end-joining. Therefore, extensive
DNAendresectioncommitsDSBrepair to theHR-mediatedpathwayandaccordingly,
theinitiationofDNAendresectionisstrictlycontrolled(SymingtonandGautier2011;
Chapmanetal.2012;Shibata2017).ThiscontrolmechanismallowsHRtobeinitiated
onlywhenarepairtemplateisavailable(S/G2phase),andthuslimitsthepotentialfor
illegitimate recombination (i.e., recombination between not fully homologous DNA
sequences).Thisiselegantlyachievedthroughtheactivationofkeyresectionfactorsby
cyclin-dependentkinase(CDK)-catalyzedphosphorylation(Iraetal.2004;Huertasetal.
2008).Itshouldbepointedoutthatthissimplemodelhasbeenchallenged,andthereis
evidencethatlimitedorevenextendedDNAendresection,occurringintheG1phase,is
involvedinthecanonicalNHEJpathway(Biehsetal.2017).Elucidatingthedetailsand
theregulationoftheseprocesseswillbeanexcitingdirectionoffutureresearch.Since
misregulation of these DSB repair pathways is believed to result in genome
rearrangementsthataretypicalinmanycancertypes,understandingtheseprocessesis
highlyrelevantforhumanhealth.
1.3End-joiningandrecombinationprocessesinvolveseveralsubpathways
BothHRandend-joiningprocessesarenotsimplelinearpathways.Bothcanbedivided
intoseveralsub-pathways,whichsignificantlydifferintermsofrepairmechanismsand
enzyme requirements. Here, we will introduce the basic principles of these repair
processes;amoredetaileddescriptionthat includes thekeyenzymaticplayerswillbe
providedinsubsequentchapters.
With regard to the end-joining mechanisms, canonical NHEJ significantly differs
fromMMEJ(Fig.3).WhereascanonicalNHEJrequiresnoorverylimitedhomology(less
than4nt)betweenthebrokenDNAmolecules,MMEJwasfoundasaDNAend-joining
event that occurs independently of the key NHEJ factors and usually involves short
stretches ofmicrohomology (2-20 nt) between the two brokenDNA ends tomediate
repair(Seoletal.2017).Therefore,MMEJissometimesconsideredaseparateprocess
thatstandsbetweentheNHEJandHRpathways.
Recombination processes, in a broad sense, can be divided into single-strand
annealing (SSA), synthesis-dependent strand annealing (SDSA), break induced
replication (BIR) and canonical HR (also called canonical DNA double-strand break
repair, DSBR) (Kowalczykowski 2015). Themain conceptual differences between the
mechanismsofthesesub-pathwaysareschematicallyillustratedinFig.4.Dependingon
whether the flanking sequences of the recombiningDNAmolecules are exchanged or
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not, recombination leads to crossover or non-crossover recombination products. A
crossover is defined as an eventwhere thedistalarmof thebrokenDNA is swapped
withthedistalarmofthetemplateDNAmolecule.AsschematicallydepictedinFig.4d,a
crossover results in the "blue" DNA molecule ultimately joined with the "red" one.
Crossovers that occur between two homologous loci of sister chromatids give rise to
"equal"sisterchromatidexchanges,whicharemutagenicallysilent(Fig.4).Incontrast,
crossovers that occur between two ectopic loci (non-homologous loci, such as in
repetitive sequences) of sister chromatids give rise to "unequal" sister chromatid
exchanges. Likewise, crossovers resulting from recombination between non-sister
(homologous) chromosomesalso lead to grossgenome rearrangements. Furthermore,
events when genetic information from one DNA molecule gets unidirectionally
transferredintoanotherDNAmoleculearereferredtoasgeneconversions.Thisoccurs
when a DNA sequence is copied from a donor template to the brokenDNAmolecule
duringBIR,SDSAorcanonicalHR.Asabove,geneconversioncanbemutagenicwhenan
ectopic site of a sister chromatid or non-sister chromosome is used as a template.
MutationsarisinginthecourseofDSBrepairbyeitherNHEJorHRmaygiverise toa
lossof heterozygosity,which is a keydriverof tumorigenesis (FearonandVogelstein
1990).
SSA involves DNA end resection to reveal repetitive DNA sequences, which are
subsequentlyannealed.TheresultingDNAflapsarecleavedandthestrandsareligated.
SSAleadsto thedeletionof theDNAsequencebetweenthe tworepeats,and is thusa
verymutagenicrepairprocess(Fig.4a).SSAisrestrictedtosituationswhentworepeats
flankthebreaksite,andcansuccessfullyrestoretheintegrityofDSBswithinrepetitive
DNA sequences. Although SSA is conceptually similar to MMEJ, it is usually grouped
togetherwithrecombinationmechanismsbecauseofenzymerequirements(seebelow).
Additionally,unlikeMMEJ,SSArequiresextensiveDNAendresection,which isshared
amongallrecombinationsub-pathways(Bhargavaetal.2016).
Inmostcases,DNAendresectionisfollowedbythe invasionof theresectedDNA
into the dsDNA template, forming a joint molecule intermediate (Kowalczykowski
2015).This initiatesDNAsynthesis,whichrestoresmissinggenetic informationat the
breaksite.InSDSA,thejointmoleculeintermediateisdestabilizedandthenascentDNA
isannealed to theotherendof thebrokenDNAmolecule(Fig.4b).SDSA isoneof the
least mutagenic recombination sub-pathways, resulting in a non-crossover repair
product. InBIR,DNA synthesisproceeds all theway to the endof the templateDNA,
copying the sequence of the entire chromosome arm (Fig. 4c). BIR is thus a unique
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pathway for repair of one-endedDSBs resulting fromcollapsedDNA replication forks
(Sakofsky and Malkova 2017). Additionally, BIR allows telomere lengthening in the
absenceoftelomerase(SakofskyandMalkova2017).ThegeneticresultofBIRisanon-
reciprocalcrossover.
In the canonical recombinational DSBR pathway, the joint molecule (D-loop)
intermediate is processed into double Holliday junctions (dHJs), which may be
processedintonon-crossoversorcrossovers,dependingonthedHJprocessingpathway
utilized (see chapter 5) (Fig. 4d) (Szostak et al. 1983). Although the various repair
events described above share some processing steps, they have different degrees of
mutagenicpotential.Todate,manyofthekeyrepairfactorsforeachpathwayhavebeen
identified;however,howacelldetermineswhichpathwaytouseforDSBrepairisstill
poorlyunderstood.TheinitialprocessingofbrokenDNAendsseemstobethekeystep
thatdetermineswhichpathwayisusedtorepairaDSB,andwillbedescribedinthenext
section.
2ProcessingofDNAbreaksforrepair
DNAendresectioninvolvesthedegradationofthe5'-terminatedDNAstrandinthe5'to
3'directionfromthebreaksitetogeneratea3'ssDNAoverhang.Generationofthis3'-
terminatedssDNAisessentialtoallowfortheusageofhomologousDNAsequencesfor
repair.ThehomologymaybeeitherbetweenthetworesectedendsofthebrokenDNA
molecule,suchasincaseofMMEJorSSA,orbetweentheresectedbrokenDNAmolecule
and an intact dsDNA template, such as in case of BIR, SDSA and canonical
recombinational DSBR. Nucleolytic resection of DNA ends typically inhibits canonical
NHEJ.
2.1ProcessingofDNAendsforcanonicalNHEJ
ManycanonicalNHEJeventsinvolvelittleornoprocessingofthebrokenDNAends.The
initialstepofNHEJinvolvesthebindingoftheDNAendsbytheKu70-80heterodimer,
whichformsaringthatencirclestheduplexDNA(GottliebandJackson1993;Ramsden
and Gellert 1998). This protects DNA ends from degradation, and recruits additional
NHEJ components (Fig. 3a). Next, the Ku70-80-bound ends are tethered by DNA-
dependent protein kinase catalytic subunit (DNA-PKcs), followed by ligation of the
broken DNA ends by the XRCC4-XLF complex and DNA Ligase IV. The yeast Mre11-
Rad50-Xrs2(MRX)complexhasastructuralroletopromoteligation,whilethefunction
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ofthehumanMRE11-RAD50-NBS1(MRN)complexinNHEJislessapparent(Chenetal.
2001;HuangandDynan2002;Zhangetal.2007;Rassetal.2009;Xieetal.2009).
InthecaseofDNAendsthatarenotdirectlyligatable,whichmayincludethosewith
DNAoverhangs,gapsorblockingchemicalgroups,limitedDNAendprocessingmaybe
required.This involves anucleolytic removal of overhangsor chemical groupsby the
humanArtemisnuclease,whichcleavesat the junctionsofsingleanddouble-stranded
DNA,andisactivatedbyDNA-PKcs(Maetal.2002;Changetal.2017;LobrichandJeggo
2017). Artemis may not be the only NHEJ nuclease; other proteins, including APLF,
Werner'ssyndromehelicase(WRN),theMRNcomplex,FEN1andEXO1mayalsoplaya
role insomecases(Changetal.2017).Alternatively, the fillingofDNAgapsatbreaks
mayfacilitateligation,whichiscarriedoutbyDNApolymerasesμandλ(Bebeneketal.
2014; Moon et al. 2014). Additionally, polynucleotide kinase (PNK) may remove 3'
phosphategroupsandphosphorylate5'OHgroups,whichmaybenecessaryforligation
(Chappelletal.2002).Finally,onerecentstudyfoundthatextendedDNAendresection
can occur prior toNHEJ in G1 (Biehs et al. 2017), but furthermechanistic analysis is
requiredtofullyunderstandthisprocess.
2.2ProcessingofDNAendforhomologousrecombination
In contrast toNHEJ, extendedDNA end resection is an obligate step that initiates all
recombinationpathways.DNAendresectionofthe5'-terminatedDNAstrandoccursin
two main steps (Mimitou and Symington 2008; Zhu et al. 2008). The first step is
catalyzed by the MRN complex and CtIP in human cells, and MRX and Sae2 in S.
cerevisiae(JohzukaandOgawa1995;KeeneyandKleckner1995;PaullandGellert1998;
Sartorietal.2007).Thenucleolyticprocessingbytheseproteinsislimitedtothevicinity
oftheDNAend(generallyupto300ntinyeast),andisthusreferredtoasshort-range
DNAend resection (Zhuet al. 2008).Themost likelymechanism for the short range-
resectionbyMRX/NisillustratedinFig.5.Resectionisinitiatedbytheendonucleolytic
cleavageof the5'-terminatedDNAstrandaway fromtheDNAend, followedby3'→5'
exonucleasethatproceedsbacktowardtheDNAend.
Both exonuclease and endonuclease activities during the first resection step are
likelycatalyzedbyMRE11/Mre11inhumanandyeastcells(Nealeetal.2005;Garciaet
al. 2011; Cannavo and Cejka 2014; Shibata et al. 2014). MRE11/Mre11 first
endonucleolytically cleaves 5' terminated DNA in the vicinity of the DNA end. This
endonucleolytic cleavage requires the ATPase activity of RAD50/Rad50 as well as
CtIP/Sae2 andNBS1(butnot strictlyXrs2 in yeast) as co-factors (CannavoandCejka
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2014; Anand et al. 2016; Deshpande et al. 2016; Oh et al. 2016; Kim et al. 2017).
Importantly, the capacity of Sae2 and CtIP to promote MRE11/Mre11 depends on
phosphorylationofkeyresiduesinCtIP/Sae2,at leastsomeofwhichareundercyclin-
dependentkinaseCDKcontrol(Huertasetal.2008;HuertasandJackson2009;Cannavo
and Cejka 2014; Anand et al. 2016; Deshpande et al. 2016). Cell-cycle dependent
phosphorylationofCtIP/Sae2representsoneofthekeycontrolmechanismsthatallow
resection(andhencerecombination)toinitiateonlyinSandG2phasesofthecellcycle
when a sister chromatid is available as a template for repair (Orthwein et al. 2015).
Downstream of the endonuclease cut, MRE11/Mre11 subsequently uses its 3'→5'
exonuclease activity to proceed back toward the DNA end, generating a 3' ssDNA
overhang. Additionally, another nuclease, EXD2, may function alongside MRE11
exonuclease in human cells (Broderick et al. 2016). It has been also proposed that
CtIP/Sae2arenucleases(Lengsfeld etal.2007;Makharashvili etal.2014;Wangetal.
2014),buttheirpotentialcatalyticfunctionsinresectionremainundefined.
The initial endonucleolytic cleavage away from theDNAendallows the resection
machinery to bypass end-binding factors or non-canonical structures that may be
presentatthebreakend.Thisincludesproteinblocks,suchasSpo11inmeiosis,stalled
topoisomerasesorKu(KeeneyandKleckner1995;Keeneyetal.1997;Nealeetal.2005;
Bonetti etal. 2010;Mimitou andSymington2010; Langerak etal. 2011;Chanut etal.
2016).Indeed,theefficiencyof5'DNAendcleavageinvitrobyMRN-CtIPorMRX-Sae2is
stimulated by the presence of protein blocks at DNA ends (Cannavo and Cejka 2014;
Anandetal.2016;Deshpandeetal.2016).Theshort-rangeDNAendresectionpathway
is absolutely required for the processing of protein-blocked DNA ends, but may be
dispensable for theresectionofcleanDNAends inyeast (Nealeetal.2005;Zhuetal.
2008;MimitouandSymington2010).Instead,bothMRE11andCtIParerequiredforall
resection events in human cells, although it remains to be definedwhether resection
alwaysdependsonthenucleaseofMRE11(Sartorietal.2007).
The initial endonucleolytic cleavage byMRE11/Mre11 creates entry sites for the
long-range resection enzymes. These subsequently catalyze resection in the 5'→3'
directionawayfromtheDNAendtogenerateextendedssDNAoverhangs(uptoseveral
kilobasesinlength)andrepresentthesecondstepoftheresectionprocess.The3'→5'
exonucleolytic DNA degradation byMRE11/Mre11 and the 5'→3'degradation by the
long-range enzymes downstream of the endonucleolytic cut has been termed "bi-
directional"resection.Inadditiontothenucleasefunction,theMRN/MRXcomplexhas
non-catalytic(i.e.structural)rolestorecruitthelong-rangeresectionenzymes(Cejkaet
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al.2010a;Nicoletteetal.2010;Niuetal.2010;Nimonkaretal.2011).Thelong-range
resectionfactorsincludeeitheroftwonucleases,EXO1/Exo1orDNA2/Dna2,whichare
well conserved between human and yeast cells (Gravel et al. 2008; Mimitou and
Symington2008;Zhuetal.2008).EXO1/Exo1isadsDNAspecificexonuclease(Tranet
al. 2002), which specifically degrades the 5'-terminated DNA strand within dsDNA,
generating 3' ssDNA overhangs. In contrast DNA2/Dna2 is an ssDNA specific 5'→3'
nuclease that cannot process dsDNA on its own, and requires a cognate RecQ family
helicasepartner(Baeetal.1998;Zhuetal.2008;Levikovaetal.2013).This includes
Sgs1 in yeast and either Bloom syndrome helicase (BLM) or WRN in human cells
(Sturzenegger et al. 2014; Pinto et al. 2016; Levikova et al. 2017). Sgs1/BLM/WRN
unwinds dsDNA to generate ssDNA, which becomes rapidly coated by replication
proteinA(RPA).RPA-coatedssDNAissubjecttodegradationbyDNA2/Dna2(Cejkaet
al. 2010a; Niu et al. 2010). RPA was found to promote 5' DNA end degradation by
DNA2/Dna2,whileatthesametimeinhibiting3'enddegradation.RPA,whichphysically
interactswith the RecQ family helicase and DNA2/Dna2, is thus a critical factor that
enforces the correct DNA polarity of DNA end resection by DNA2/Dna2 (Cejka et al.
2010a;Niuetal.2010).BothhumanDNA2andyeastDna2,inadditiontotheiressential
nuclease activity, contain a helicase domain, which likely functions as an ssDNA
translocase to facilitate the degradation of 5'-overhanged DNA by the DNA2/Dna2
nuclease(Levikovaetal.2017;Milleretal.2017).TheindividualsubunitsoftheSgs1-
Dna2-RPA,BLM-DNA2-RPAandWRN-DNA2-RPA complexes stimulate theactivities of
theirpartnerstoformintegratedmolecularresectionmachines(Cejkaetal.2010a;Niu
etal.2010;Pintoetal.2016).
MultipleDNAendresectionmechanismsdescribedabove leadto the formationof
3’-tailed ssDNAcoatedbyRPA.Thekey functionofRPA is toprotect ssDNA from the
actionofnucleasesandpreventtheformationofsecondarystructuresthatmightarise
byself-annealingofssDNA(Wold1997).Aswillbedescribed in thesectionbelow, in
SDSA,BIRandcanonical recombinationalDSBRpathways,RPAmustbereplacedwith
the strand exchange protein RAD51. In contrast, SSA shares the initial DNA end
resectionwithother recombination sub-pathways, but isRAD51-independent. Instead
of using intact dsDNA as a template, SSA functions by annealing the two resected
strandsofDNA,using large stretchesof sequencehomology to forma stable complex
between the two resected broken DNA molecules (Fig. 4a). This is dependent on
RAD52/Rad52,whichhasacapacitytoannealRPA-coatedssDNA.Afterannealing,the
non-complementary sequences are cleavedbyXPF-ERCC1 (Rad1-Rad10 in yeast) and
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anyremaininggapsarefilledandligatedtocompletetherepairoftheDSB(Bardwellet
al.1994;Ivanovetal.1996;Mortensenetal.1996;Shinoharaetal.1998).
Broken DNA molecules signal the presence of DNA damage to the cellular
checkpointmachinery.Generally,DSBsactivatetheATM(Tel1inyeast)kinase.Inboth
systems,MRN/MRX plays a structural role to activate ATM/Tel1 (Carson et al. 2003;
Uzieletal.2003;LeeandPaull2004;LeeandPaull2005).Uponresection,RPA-coated
ssDNA thenactivates theATR-ATRIP(Mec1-Ddc2 in yeast)pathway (ZouandElledge
2003).ATM/Tel1andATR/Mec1sensorsphosphorylatehundredsofproteintargetsat
SQ/TQ motifs, which activate the proper response to DNA damage. This includes
regulation of DNA repair components and checkpoint proteins, leading to cell cycle
arrest and thus providing time for repair (Jackson and Bartek 2009). Additionally,
unsuccessfulrepairandprolongedcellcyclearrestleadtotheactivationofapoptosisin
highereukaryotes(JacksonandBartek2009).
2.3ProcessingofDNAendsforAlt-EJ(MMEJ)
IntheabsenceofthekeycanonicalNHEJfactors,includingKu70-80,DNA-PKcs,XRCC4,
XLFandDNALigaseIV,cellscanstillrepairDSBsthroughanend-joiningeventreferred
toasMMEJ(SimsekandJasin2010;Changetal.2017)(Fig.3b).ThefrequencyofMMEJ
in DSB repair is not very clear yet (Sfeir and Symington 2015), but it has been
establishedthattheprocessismorecommoninhumancellscomparedtoyeast.Oneof
thehallmarksofMMEJistheuseofsharedsequencemicrohomologybetweenthebreak
points,whichisgenerallylimitedto~2-20ntinlength(Changetal.2017).Overlapping
homologies were observed in junction points upon translocations in human cancers
(Stephensetal.2009),suggestingthattheymayariseduetoMMEJ.Currently,onlyafew
factorshavebeenimplicatedinmediatinghumanMMEJ,includingFANCA,PARP1,DNA
LigaseIII,CtIP,DNA2andPolθ(Poltheta,alsoknownasPolQ),butnoneoftheseappear
tobeabsolutelyessentialforMMEJ(Audebertetal.2004;Bennardoetal.2008;Simsek
etal.2011;Howardetal.2015;Mateos-Gomezetal.2015;Mateos-Gomezetal.2017).
The use of microhomology for repair indicates that MMEJ and recombination-based
mechanisms may share elements of DNA end resection. This is also supported by
observationsthatKu70-80inhibitsMMEJ,andKuisknowntobeinhibitoryforDNAend
resection(MimitouandSymington2010).Theendresection factorsCtIPandDNA2 in
humancellshavebeenidentifiedasbeingimportanttopromoteMMEJeventsbutit is
stillunclearifthisisduetoadirectroleinDNAendresection(ZhangandJasin2011;
Howardetal.2015).Likewise,thegeneticrequirementsforMMEJremaintobedefined
13
in yeast. Interestingly, MMEJ was increased in mre11, sae2 and sgs1 exo1 mutants,
indicatingthattheabsenceofthecanonicalDNAendresectionfactorsisnotlimitingfor
MMEJ(Wangetal.2006;Dengetal.2014).Whetheranotheryetunidentifiednucleaseis
essentialforMMEJisnotclear.RPAisclearlyastronginhibitorofMMEJinbothyeast
andhumancells(Dengetal.2014;Mateos-Gomezetal.2017).Thisshowsthatthemore
extensiveresectioncreatingssDNAoflengthsthatstronglyassociatewithRPAchannels
repair towardHR. Inaccord, themotoractivityof humanPolθwas foundtodisplace
RPAtopromoteMMEJ,showingthatthebalancebetweenHRandMMEJiscontrolledby
theopposingactivitiesofPolθandRPA(Mateos-Gomezetal.2017).
3FormationofRAD51-ssDNAfilamentonresectedDNA
Resection of the 5’-terminated DNA strand at DSBs leads to the formation of 3’-
overhanged ssDNA, which is initially coated by RPA. In a subsequent step, RPA is
replacedbythekeyrecombinationproteinRAD51(Rad51inyeast)(Fig.6).RAD51and
ssDNAformanucleoproteinfilament,alsocalledapre-synapticfilament.Thiscatalyzes
the signature step of the recombination pathway, which includes homology search,
pairingwiththeintactdonor(alsocalledtemplate)dsDNAandstrandinvasion(Benson
etal.1994;Sugiyamaetal.1997).ThetransientinteractionoftheRAD51nucleoprotein
filamentwiththetemplatedsDNAisreferredtoasasynapticcomplex.Thisultimately
leads to thedisplacementofoneof the templateDNAstrands formingadisplacement
loop (D-loop, also called joint molecule) intermediate (Fig. 6), which represents the
post-synapticstage.Allthesestepsarecontrolled(bothpositivelyandnegatively)bya
numberof recombination regulators,whichoften physically interactwithRAD51and
allowrecombinationtooccuronlyinthepropercontext.
TounderstandmechanismsunderlyingtheregulationofRAD51,it is importantto
understandthatRAD51hasacapacity tobindbothsingle-anddouble-strandedDNA,
butonlyssDNAbindingisthoughttopromoterecombination,whiledsDNAbindingby
RAD51 is generally inhibitory (Zaitseva et al. 1999). Furthermore, RAD51 binds and
hydrolyzesATP.ATPbinding,butnotATPhydrolysis,byRAD51isrequired forstable
RAD51-DNA binding; in contrast, ATP hydrolysis by RAD51 is required for the steps
downstream of DNA invasion (Sung 1994; Baumann et al. 1996; vanMameren et al.
2009).RAD51canalsohydrolyzeATPnon-productively(i.e.,withoutcatalyzingstrand
invasion);also inthiscase,ATPhydrolysis leads toareduction in itscapacity tobind
DNA. Recombination can be stimulated on several levels by affecting these RAD51
activities.ThisincludesRAD51loadingonRPA-coatedssDNA(i.e.exchangeofRPAwith
RAD51), reduction of RAD51's capacity to bind dsDNA, stabilization of the nascent
14
nucleoproteinfilamentbyinhibitingitsdisassembly,aswellasremodelingoftheRAD51
nucleoproteinfilamentintoaconformationthatisoptimallypermissiveforDNAstrand
exchange. These control mechanisms are in place to prevent aberrant recombination
(Heyer2015).Thisincludesrecombinationbetweennon-homologoussequences,which
mayleadtoDNAtranslocationsandgenomerearrangements,orrecombinationduring
physiological processes of DNA metabolism when ssDNA is present, such as during
unperturbedDNAreplicationortranscription.Theproperinterplayofbothpositiveand
negativerecombinationregulatorsascertainsthatrecombinationoccursonlywhenitis
neededtooptimallymaintaingenomestability(Heyer2015).
The first of the control mechanisms is the replacement of RPA with RAD51 on
resected ssDNA. Due to the higher affinity toward ssDNA, RPA initially outcompetes
RAD51forssDNAbinding.ToovercomethisapparentinhibitoryeffectofRPA,cellshave
variousrecombinationmediatorproteinsthathelploadRAD51ontossDNA,displacing
RPA in theprocess. Inyeast,thekeyrecombinationmediator thatdisplacesRPA from
ssDNA is Rad52. The interaction of Rad51 with Rad52 is required for this process
(Benson et al. 1998; Song and Sung 2000). Human RAD52, in contrast, possesses no
recombinationmediatoractivity,anditscontributiontorecombinationinhumancellsis
much more subtle than in yeast, while Caenorhabditis elegans and Drosophila
melanogaster lackRAD52.Themainmediatorprotein inhigher eukaryotes, including
worms, flies and humans, is the product of the breast cancer susceptibility gene 2
(BRCA2)anditshomologues(Yangetal.2005;Petalcorinetal.2007;Jensenetal.2010).
DepletionofBRCA2incellstreatedwithionizingradiation(IR)leadstopersistentRPA
anddecreasedRAD51fociformationonbrokenDNA,clearlyindicatingitsroleinRAD51
loading(Yuanetal.1999).BRCA2containseightBRCrepeats(conservedmotifsofabout
35aminoacids),whichcanallindependentlyinteractwithRAD51,althoughonlyfourto
five repeats associatewith RAD51 at a given time (Carreira et al. 2009; Jensen et al.
2010).Therepeats1-4bindRAD51withahighaffinityandpromoteRAD51nucleation
on ssDNA; repeats 5-8 show a lower affinity to free RAD51 and rather stimulate the
growthofthenascentRAD51filament(Jensenetal.2010;Liuetal.2010;Thorslundet
al. 2010). Mechanistically, the BRC repeats 1-4 of BRCA2 promote ssDNA binding of
RAD51 by inhibiting its ATPase activity, which stabilizes ssDNA binding of RAD51.
Additionally, BRCA2 promotes recombination by inhibiting RAD51's dsDNA binding
activity(Jensenetal.2010).ThedisplacementofRPAfromssDNAbyBRCA2isfurther
facilitatedbyDSS1,adirectinteractionpartnerofBRCA2(Yangetal.2002;Zhaoetal.
2015). BRCA2 also interacts with PALB2, which was shown to promote RAD51-
mediated DNA strand exchange on its own (Buisson et al. 2010). In cells, PALB2
15
mediates BRCA2's recruitment to DNA damage and bridges BRCA2's interactionwith
BRCA1(Syetal.2009).Cellularassaysestablishedthattheseinteractionsarecriticalfor
recombination, although the mechanisms how BRCA1 and PALB2 proteins affect
BRCA2's recombinationmediator activity remain to be defined. RAD51 nucleoprotein
filament assembly is also stimulated by RAD54, independently of RAD54's ATPase
activity(WolnerandPeterson2005).AdditionalproteinsincludingtheMMS22L-TONSL
complexmay promote the RAD51 nucleoprotein filament assembly during perturbed
DNAreplication(Piwkoetal.2016).
The activity of RAD51 is also promoted by a group of proteins termed RAD51
paralogs.Therespectivegeneslikelyaroseduringevolutionthroughaduplicationofthe
RAD51 gene, and share around 20 to 30% of sequence homology with RAD51. The
paralogs are represented by five polypeptides in human cells: RAD51B, RAD51C,
RAD51D, XRCC2 and XRCC3, which form two major complexes: RAD51B-RAD51C-
RAD51D-XRCC2(BCDX2)andRAD51C-XRCC3(CX3)(Massonetal.2001).Neitherofthe
paralogproteinsnorcomplexesexhibitsDNAstrandexchangeactivityontheirown,and
all likely function via regulatingRAD51. Phenotypically, depletion of these complexes
generallyresultsinfewerRAD51fociinresponsetoionizingradiation.Thisresembles,
althoughtoalesserextent,thedepletionofBRCA2.Theparalogslikelyfunctioninthe
samepathwayasBRCA2as indicatedby epistatic interactions(Qing et al. 2011).The
first mechanistic report indicated that the RAD51B-RAD51C proteins, similarly to
BRCA2,havearecombinationmediatoractivitytofacilitateloadingofRAD51onRPA-
coated ssDNA, displacing RPA in the process (Sigurdsson et al. 2001). In yeast, the
RAD51paralogsincludeRad55andRad57(Sung1997;Liuetal.2011a).Rad55-Rad57
physically interact with the Shu complex, comprising of Csm2, Psy3, Shu1 and Shu2
polypeptides,andRad52.Thesubunitsofthesupercomplexsynergizeintheircapacity
todisplaceRPAandfacilitateRad51-ssDNAnucleoproteinfilamentformation(Gaineset
al.2015).ThehumanorthologofShu2 isSWS1,which formsacomplexwithSWSAP1
(Martin et al. 2006; Liu et al. 2011b).TheheterodimerbindsDNAand interactswith
RAD51,RAD51DandXRCC2. DepletionofSWS1resultsinareductionofspontaneous
and radiation-induced RAD51 foci. This suggests that SWS1 in humans also likely
functions inconjunctionwith theRAD51paralogs,but their interplay,aswellas their
relationshiptoBRCA2'sfunction,remainundefined.EvidencefromtheC.elegansmodel
system instead suggests that the RAD51 paralogs, represented by RFS-1 and RIP-1,
function downstream of BRCA2, specifically in the stabilization of the RAD51
nucleoprotein filament and its remodeling into a species that is optimally capable to
16
invadetemplatedsDNA(Tayloretal.2015;Tayloretal.2016).RAD51filamentsarealso
stabilizedbytheSWI5-SFR1complex(AkamatsuandJasin2010;Tsaietal.2012).
It is well established that there is a balance between factors that promote the
formation and disruptRAD51/Rad51 nucleoprotein filaments (Heyer 2015). In yeast,
Srs2candismantleRad51 filamentsdue to itsATP-poweredDNAtranslocaseactivity,
andadditionallyviastimulatingtheATPhydrolysisofRad51throughadirectphysical
interaction,whichdestabilizesthenucleoproteinfilament(Krejcietal.2003;Veauteet
al.2003;Liuetal.2011a).Inhumans,nodirecthomologofSrs2hasbeenidentified,yet
themostprobablecandidatesforthisfunctionareRECQ5,FBH1,PARIorBLMhelicases
(Bugreev et al. 2007b; Hu et al. 2007; Fugger et al. 2009; Schwendener et al. 2010;
Moldovan et al. 2012; Patel et al. 2017). To elucidate the functional interplay of the
various RAD51 regulators represents a challenge for future research. The proper
balance between pro- and anti-recombination factors is required to execute
recombinationonlywhenitisneededandthuspreventillegitimaterecombinationand
genomerearrangements.
4HomologysearchandDNAstrandexchange
Once the pre-synaptic filament is formed and stabilized, it begins the search for a
homologoussequence.Inmostcasesinvegetativecells,thesisterchromatidisusedasa
repairtemplate.ArecentreportdemonstratedthatthehumanBRCA1protein,whichis
similarlyasBRCA2atumorsuppressorthatisfrequentlymutatedinfamiliarbreastand
ovariancancers,directlypromotesDNAinvasion.Thislikelyoccursthroughpromoting
the assembly of the synaptic complex or promoting the homology search (Zhao et al.
2017).Despitethecomplexnuclearenvironment,thecourseofhomologysearch–the
processthatoccursimmediatelybeforethesynapticphase–isrelativelyfast.Inyeast,it
has been demonstrated that the mobility of a cut chromosome is increased, which
allowstheRad51filamentstoexplorealargernuclearvolume(Dionetal.2012;Mine-
Hattab and Rothstein 2012). Additionally, broken DNA relocalizes to the nuclear
periphery(Ozaetal.2009;Chioloetal.2011;Horigomeetal.2014;Ryuetal.2015).
Althoughtheexactmechanismofhomologysearchisstillundefined,itissuggestedthat
thepre-synapticfilamentrandomlyprobesthegenomebymakingmultipletemporary
contactswithdifferentDNAduplexes(ForgetandKowalczykowski2012;Renkawitzet
al. 2014; Qi et al. 2015). Contacts with very short microhomologies are unstable;
instead, contactswithmore than7nt of homology aremore stable,which allows the
presynaptic complex to probe flanking sequences for additional homology. Once
17
homologyisidentified,thepre-synapticfilamentinvadestheduplexDNA,displacesthe
originalstrandandbinds itscomplementarysequencebyWatson-Crickpairing. Ithas
been estimated that the efficiency of repair in yeast was decreased to ~14% when
templatesequencedivergedbyaboutoneineveryeightnucleotides(Anandetal.2017).
Followingstrandinvasion,thedisplacedssDNAwithintheD-loopstructureisstabilized
byRPA,whichpreventsreversaloftheD-loopformation(LaveryandKowalczykowski
1992;Eggleretal.2002).Additionally,RAD51isremovedfromtheheteroduplexdsDNA
oftheD-loopstructure.ThisfunctioniscatalyzedbyRAD54,adsDNAtranslocasethat
usesitsATPase-poweredmotoractivitytodisplaceRAD51fromdsDNA(Solingeretal.
2002;Masonetal.2015)(Fig.6).RAD54thuspromotesD-loopstability,whichallows
DNAsynthesisandfacilitatescanonicalHR(CeballosandHeyer2011;WrightandHeyer
2014).
The 3'-terminated strands within D-loop structures that resist disassembly can
prime DNA synthesis. Although translesion polymerases have been implicated in HR
(Kawamoto et al. 2005; McIlwraith et al. 2005), most of DNA synthesis during
recombinationislikelycatalyzedbyeitherpolymeraseδorpolymeraseε(Lietal.2009;
Hicksetal.2010;Wilsonetal.2013).However,theDNAsynthesisduringrecombination
is about three orders of magnitude more error-prone than in DNA replication, most
likelyduetolimitedactivityofDNAmismatchrepairduringrecombination-dependent
repairDNAsynthesis(Hicksetal.2010).DNAsynthesisextendsthelengthofthepaired
duplex, which further stabilizes the joint molecule, leading to the recovery of the
missinggeneticinformationinthebrokenDNAmoleculebyusingtheinvadedmolecule
as a template. Downstream of strand invasion and DNA synthesis, recombination
proceedsintoeitherofthreerecombinationsub-pathways:BIR,SDSAandcanonicalHR
(see chapter 1.3 for general introduction to these pathways and Fig. 4). In BIR, the
invadedDNAmoleculeisstabilized,andDNAsynthesisproceedsalongthewholelength
ofthetemplateDNAviaabubble-likestructuretothechromosomeend(Llorenteetal.
2008;SakofskyandMalkova2017).TheextendedDNAsynthesisislikelycatalyzedby
polymerase δ, in conjunction with the Pif1 helicase, which promotes the strand
displacementactivityofpolymeraseδ(Wilsonetal.2013).Interestingly,DNAsynthesis
during BIR occurs conservatively, and thus dramatically differs from canonical DNA
replication (Donnianni and Symington 2013). How exactly the synthesis of the
complementarystrand isachieved remainsundefined.TheBIRpathwayoccurs at the
cost of elevated mutagenesis, and is thus primarily utilized when no alternative is
available,suchasintheabsenceofthesecondDNAend.
18
InSDSA, theextended invadedDNAstrandseparates fromthe templateDNA,and
annealstothesecondendofthebrokenDNAmolecule.DNAsynthesisandligationthen
completetherepairprocess.IncanonicalHRinstead,throughaprocesstermedsecond
DNAendcapture,thesecondbrokenDNAendannealstothedisplacedssDNAstrandof
theD-loopstructure(Fig.4).ThestrandannealingemploystheRad52proteininyeast
(Nimonkar et al. 2009). Although humanRAD52 also possesses the strand annealing
activity(McIlwraithandWest2008;Jensenetal.2010),theeffectsofRAD52-deficiency
are modest and are particularly revealed only in the absence of BRCA2 (Feng et al.
2011).Thisraisesquestionswhetherthesecondendcaptureinhumancellsismediated
byannealingorasecondDNAstrandinvasionevent(Kowalczykowski2015).
The balance between SDSA and canonical recombinational DSBR is regulated by
activitiesthateitherdisruptorpromotestabilityoftheD-loopstructure.Inmostcases,
thestabilityofD-loopsisdictatedbymotorproteinssuchasDNAhelicasesthatactby
moving the junctions.Mph1 inyeastcellsandBLM,RECQ1andRTEL1 inhumancells
have been implicated in D-loop dissociation and promotion of SDSA (Bugreev et al.
2007b;Barberetal.2008;Bugreevetal.2008;Prakashetal.2009;Daleyetal.2013;
Mitchel et al. 2013). In yeast, D-loops can also be disrupted or "dissolved" by an
alternativemechanismthatinvolvestopoisomeraseIII(Top3),whichisalsothoughtto
promoteSDSA(Faschingetal.2015).Whetherthisprocessalsofunctionsinhumancells
isnotyetclear.Additionally,RAD54'sbranchmigrationactivityhasbeenimplicatedto
disruptD-loopsdownstreamofRAD51-mediatedstrandinvasioninvitro(Bugreevetal.
2007a), but the biological significance of this function may be restricted to limiting
recombinationwithnon-homologousDNA(CeballosandHeyer2011;WrightandHeyer
2014). Regulating the balance between SDSA and HR is important, as it affects the
geneticoutcomeofrecombination.WhereasSDSAonlyleadstonon-crossoverproducts,
canonicalHRisapathwaythatcanpotentiallyproducecrossovers,aswillbedescribed
inthenextchapter.
5Processingrecombinationintermediates
D-loop stability determines the pathway choice between SDSA and canonical
recombinational DSBR. In SDSA, the D-loop is disrupted, whereas in recombinational
DSBR, theD-loop isstabilizedandbecomesasubstrate forannealingwith thesecond
resecteddsDNAend(Fig.4).Thisgivesrisetoa"double"or"complement-stabilized"D-
loop.ThisstructureformsasaresultofannealingactivitycatalyzedbyRad52inyeast,
and doesnot represent a second strand invasion step (Nimonkar et al. 2009). Rad52
19
wasshowntopromoteannealingofssDNAtoD-loopsgeneratedbycognateRad51and
Rad54inthepresenceofRPA,indicatingthattheprocesslikelyrequiresdirectprotein-
proteininteractions.ThisfunctionofRad52isapparentinunicellulareukaryotes,while
RAD52inhumancellshasamuchlessdefinedfunction(Fengetal.2011).Itispossible
thatinhigheukaryotesotherproteinssuchasBRCA2mightbeinvolvedinsecondend
captureinadditiontoRAD52.Itremainstobeestablishedwhethersecondendcapture
inhumancellsemploysannealingand/orstrandinvasionmechanisms.
Following second end capture, DNA synthesis and ligation gives rise to a central
intermediate of canonical HR, termed a double Holliday junction (dHJ)(Duckett et al.
1988).AsbothDNAmoleculesarephysicallylinkedatthejunctionpoints,HJsneedto
beprocessedpriortoseparationofbothDNAmolecules(Fig.7).AfailuretoprocessHJs
leads to chromosome segregation defects, and may be one of the mechanisms
responsibleforgenomeinstability(Wechsleretal.2011).Duetothehomologybetween
therecombiningDNAmolecules,akeyfeatureofendogenousHJsistheirmobility:the
junctionpointscanmoveineitherdirectiontoalimitedextentspontaneously,ormore
extensively inATP-hydrolysisdriven reactions catalyzedbymolecularmachines, such
as DNA helicases or translocases. Double HJs can be processed by resolution or
dissolution-based mechanisms. As will be described below, these processes are
enzymaticallydistinctandleadtodiversegeneticoutcomes.Whereasdissolutionleads
tonon-crossoverproducts,resolutiongivesrisetobothcrossoversandnon-crossovers
(Fig.4andFig.7).
5.1DissolutionofdoubleHollidayjunctions
DissolutionseparatestherecombiningDNAmoleculeswithoutexchangingtheflanking
sequences. As somatic cells employ mechanisms to maximally preserve genome
integrity, dissolution is the default mode of dHJ processing. In yeast, dissolution is
carriedoutbytheSgs1-Top3-Rmi1(STR)complex(Cejkaetal.2010b),whileinhuman
cells the"dissolvasome"consistsofBLM, topoisomerase IIIαandRMI1-RMI2, forming
theBTRRcomplex(WuandHickson2003;Singhetal.2008;Xueetal.2013).AsSgs1,
BLM can unwind various DNA structures and branch migrate HJs in vitro (Wu and
Hickson 2003). The dissolution reaction involves migration of the two Holliday
junctionstowardeachother(i.e.convergentbranchmigration)bythecombinedactivity
of the RecQ family BLM/Sgs1 helicase and the strand passage activity of the
TOPOIIIα/Top3 type IA topoisomerase (Fig. 7a). As both Holliday junctions link two
DNAmoleculesoflonglengths,theendsarenotfreetorotate,andthedHJstructureis
20
topologically constrained. Therefore, a branch migration activity of Sgs1/BLM is not
sufficienttomigrateendogenousHJs(Cejkaetal.2010b;Chenetal.2014).Ithasbeen
demonstratedthatthestrandpassageactivityofTOPOIIIα/Top3isrequiredtorelieve
positive supercoiling thatwouldotherwise formaheadof (between) the convergently
migratingjunctionsandpreventfurthermovement(Chenetal.2014).Mechanistically,
Top3/TOPOIIIαcreatesatransientnickinssDNAthatallowstheotherssDNAstrandto
passthroughit,therebyallowingtherelaxationofthetorsionalstressformingbetween
the junctions during convergent branchmigration. RMI1/Rmi1 does not significantly
affect the initial DNA branchmigration step, but seems to specificallypromote a late
stepjustpriortodissolution(Cejkaetal.2010b;Bocquetetal.2014).Thelastpredicted
intermediate of convergent branch migration is a hemicatenane: this structure
representsajunctionbetweentwodsDNAmolecules,whereonestrandofoneduplexis
wrapped around another strand from the second DNA duplex (Fig. 7a). It has been
proposed that RMI1/Rmi1 specifically promotes the processing of a hemicatenane
(Cejka et al. 2012). RMI2,which is onlypresent inhigh eukaryotes, likelyhasonly a
minor function indHJdissolution,andmayhaveother roles such as to targetBLMto
blocked DNA replication forks (Singh et al. 2008). Finally RPA, which physically
interactswith BLM/Sgs1 and RMI1/Rmi1 subunits of the STR/BTRR complexes, also
stimulatesdissolution(Cejkaetal.2010b;Xueetal.2013).ThislikelystemsfromRPA's
capacity to promote the helicase of BLM/Sgs1 and strand passage of TOPOIIIα-
RMI1/Top3-Rmi1, likelythroughitsssDNAbindingactivitytopreventre-annealing,as
wellasbecauseofthedirectphysicalinteractionofRPAwithRMI1/Rmi1(Broshetal.
2000;Planketal.2006;Cejkaetal.2012;Xueetal.2013).TheuniquemechanismofdHJ
processingbydissolutionexclusivelyresultsinnon-crossoverevents,whichmaintains
genome stability in vegetative cells (Fig. 7a). BLM-deficient cells are characterized by
elevated levels of sister chromatid exchanges, which are a hallmark of genome
rearrangements and may contribute to cancer predisposition of Bloom syndrome
patients (Chaganti et al. 1974). These rearrangements result from elevated usage of
pathways thatare alternative todissolutionand thataredependenton the structure-
specificnucleasesdescribedinthenextchapter.
5.2ResolutionofdoubleHollidayjunctions
DoubleHJs thatevadedissolutionareresolvedbystructurespecificnucleases later in
thecellcycletogiverisetobothcrossoverandnon-crossoverrecombinationproducts.
Nucleolytic cleavage also represents the only option for the processing of singleHJs.
21
Thesemay form duringDSB repairwhere only one strand invades template DNA, or
whenoneoftheD-looparmshasbeencleavedpriortosecondendcapture(Wechsleret
al.2011;ShahPunataretal.2017).Todate,threestructure-specificnucleasescapableof
processingHJsorsimilarstructureshavebeen identified ineukaryoticcells, including
humanMUS81-EME1andyeastMus81-Mms4,humanSLX1-SLX4andyeastSlx1-Slx4as
well as human GEN1 and yeast Yen1 (Dehe and Gaillard 2017). The Mus81-Mms4
complex in yeast was found to be essential for viability in cells lacking Sgs1-Top3,
indicating its role in the processing of recombination intermediates thatwould have
otherwise been processed by the Sgs1-Top3 complex (Hickson andMankouri 2011).
Mus81belongstotheXPFfamilyofnucleasesandrepresentsthecatalyticsubunitofthe
heterodimer. Both yeast and humanMus81-Mms4/MUS81-EME1 complexes prefer to
cleavereplicationforks,3’flapsandnickedHJs(Gaillardetal.2003;EhmsenandHeyer
2008). MUS81-EME1/Mus81-Mms4 cleave intact HJs inefficiently in an asymmetric
manner by introducing a nick a few nucleotides away from the junction, producing
gapped and flapped DNA products, which are unsuitable for ligation and necessitate
furtherprocessing(Wyattetal.2013). Inhumancells,MUS81 formsanothercomplex
withEME2,whichhasabroadersubstratespecificitythanMUS81-EME1,andhasroles
in replication fork restart (Pepe andWest 2014b). SLX1/Slx1 belongs to the GYI-YIG
familyofnucleasesandisthecatalyticsubunitoftheSLX1-SLX4heterodimer.TheSLX1-
SLX4/Slx1-Slx4 complex can cleave branched structures with a preference toward Y-
structures,5' flapsandreplication forks.While itcancleave intactHJs, thecleavage is
inefficient,asymmetrical and createspoorly ligatableproducts (Fricke andBrill2003;
Fekairi et al. 2009; Svendsen et al. 2009;Wyatt et al. 2013). BothMUS81 and SLX1
complexes are thus non-canonical HJ resolvases. The only bona-fide eukaryotic HJ
resolvase is GEN1/Yen1,which belongs to the RAD2/XPG family of nucleases. As the
EscherichiacoliRuvC,GEN1/Yen1dimerizesonHJs,andcleavesHJsbyadualincision
mechanismthatproducestwoligatableproducts(Dunderdaleetal.1991;Ipetal.2008;
Rassetal.2010).However,bothGEN1andYen1arecapableofcleavingotherbranched
DNA structures as well. The random nature of HJ cleavage by the various nuclease
complexes gives rise to both crossover and non-crossover recombination products
(ShahPunataretal.2017)(Fig.7).
Inhumancells,boththeSLX1-SLX4andMUS81-EME1dimersassociatetoformthe
SLX1-SLX4-MUS81-EME1 tetramer(SMcomplex).The formationof the SMcomplex is
mediatedbytheinteractionbetweenMUS81andSLX4polypeptides(Wyattetal.2013).
Biochemical studies demonstrated that the SM complex exhibits higher HJ resolution
activitycomparedtoitsindividualsubunits.Mechanistically,SLX1-SLX4makesthefirst
22
nick in the HJ creating a substrate for MUS81-EME1, which then makes a second
counter-nick in the opposing strand, allowing for efficientHJ resolution (Castor et al.
2013;Wyatt et al. 2013).This activity is further enhancedby interactionwitha third
nucleasecomplex,theXPF-ERCC1dimer,whichhasastructural(i.e.non-catalytic)role
to promoteHJ cleavage (Wyatt et al. 2017)(Fig. 7b). The catalytic subunitwithin the
XPF–ERCC1dimerisXPF,whichhasestablishedfunctionsinnucleotideexcisionrepair.
It should be pointed out that the functions ofMUS81-EME1/Mus81-Mms4and SLX1-
SLX4/Slx1-Slx4arenotspecifictoprocessingrecombinationproducts,andbothenzyme
complexesmaycleavestructuresarisingduringotherDNArepairprocesses,aswellas
during replication or telomere maintenance. Likewise, the SLX1-SLX4-MUS81-EME1-
XPF-ERCC1 (SMX) trinuclease complex has a broad substrate specificity that is not
restricted to HJ cleavage and likely functions in multiple DNA metabolic pathways
(Wyattetal.2017).Theformationofthenucleasecomplexesappearsspecifictohigher
eukaryotes,asnosuchcollaborativeactionofnucleaseshasbeenobservedinyeast.
5.3RegulationofHollidayjunctionprocessing
To maximally preserve genome stability, vegetative cells evolved mechanisms that
facilitatedissolutionpathwayusageoverresolution,which limitscrossover formation.
Additionally,theactivityofthenucleasescapableofcleavingbranchedDNAstructures
must be carefully controlled to avoid promiscuous DNA cleavage. The preferential
employmentofdissolutionoverresolutionbystructure-specificnucleases isgoverned
bytightspatialandtemporalcontrol(MatosandWest2014).TheSTR/BTRRcomplexis
likelyactiveinanyphaseofthecellcycle,andthusprocessesthemajorityofdHJsfrom
S-phasetomitosisinbothyeastandhumancells.TheactivityofyeastMus81-Mms4is
insteadlowinS-phase,andbecomeselevatedattheonsetofmitosisbyphosphorylation
oftheMms4subunitbyCDKandCdc5(Matosetal.2011;Matosetal.2013).Inhuman
cells, the phosphorylation of EME1 does not activate MUS81 directly, but rather
promotes the formationof the SLX1-SLX4-MUS81-EME1 (SM) complexwith increased
activitytowardHJs(Wyattetal.2013).YeastYen1isinactiveinitsphosphorylatedstate
inSphase,andbecomesactivatedbyCdc14-mediateddephosphorylationlateinmitosis.
Interestingly,phosphorylationofYen1notonlyinhibitsitscatalyticactivitybylimiting
itsassociationwithDNA,butalsopreventsnuclearimport(Kosugietal.2009;Blancoet
al.2014).Nuclearexclusionappearstobetheprimarymechanismthatrestrictshuman
GEN1 activity (Chan and West 2014). The nuclear export sequence within GEN1
ascertains thatGEN1canprocessrecombination intermediatesonlywhenthenuclear
23
envelope breaks down during mitosis. These regulatory mechanisms collectively
ascertainthatthestructure-specificnucleasesareonlyactivatedlateinthecellcycleto
removeany residual junctions thatwerenotprocessedby thedissolvasome complex.
Althoughpotentiallymutagenic,efficientprocessingofalljointmoleculesbyresolution
mechanismsisessentialtopreventchromosomemissegregationinmitosis(Wechsleret
al.2011).
6SpecializedrolesofDSBrepairpathways
CellsevolvedmechanismstorepairaccidentalDNAbreakstoachievemaximalefficiency
and accuracy in the maintenance of genome integrity. However, not all DSBs are
pathological,andthereareseveralcaseswhenDSBsareintroduceddeliberately,which
serves specific physiological purposes. The best examples are processes occurring
during lymphocyte development and inmeiosis. Aswill be seen below, during these
events cellsmakeuseof theDSB repairpathways to insteadgeneratediversity.Both
processesrepresentfascinatingexamplesoftheplasticityoftheDSBrepairsystems.
6.1DSBrepairpathwaysintheimmunesystem
V(D)JrecombinationisaprocessthatoccursduringBandTlymphocytedevelopment
andinvolvesarandomrearrangementofthevariable(V),diversity(D)andjoining(J)
segments of immunoglobulin genes (Arya and Bassing 2017) (Fig. 8a). The random
assemblyof theseVDJ segmentsallowsproducingawidevarietyof antigen receptors
from a limited number of gene segments. Despite its name, V(D)J recombination is
facilitatedbycanonicalNHEJfactors,anddoesnotinvolvehomologousrecombination.
DisruptionofkeyNHEJfactorsresultsinsevereimmunedisordersinhumansandmice,
indicating theiressentialrole inV(D)Jrecombination.TheMRNcomplexwas foundto
haveafunctioninV(D)Jrecombination,butthisappearstobedependentonitscapacity
topromotecheckpointsignalingviaactivationoftheATMkinase,andnotaroleinHR
(Uzieletal.2003;Helminketal.2009).
V(D)J recombination is initiated by the recombination-activating genes 1 and 2
(RAG1 and RAG2), which are expressed in developing lymphocytes and therefore
restrict this process to these cells (McBlane et al. 1995; Schatz and Swanson 2011).
RAGs bind to the recombination signal sequences (RSS) flanking the V, D, and J gene
segmentsandfirstcreateanssDNAnickatthejunctionbetweenthecodingregionand
theRSS.TherearetwotypesofRSsequences,whichdifferwithrespecttothelengthofa
spacerregionbetweenthetwoidenticalsequences.Thespacersareof12or23bpsin
24
length, giving rise to RS12 and RS23 sequences (Fig. 8a). Next, RAGs catalyze the
formationofapairedcomplex,wheretheRS12andRS23sequencesassociatewiththe
same RAG complex. The free hydroxyl group of the nicked strand then invades the
phosphodiester bond of the intact strand, generating a DSBwithdifferent DNA ends.
ThecodingendoftheDSBcontainsahairpinloop,whereasthesignalendisblunt.Next,
the blunt signal ends are ligated together to form a circular piece of DNA that is lost
duringsubsequentcelldivisions.Thehairpincodingendsareprocessedandjoinedby
theNHEJmachinerytofusetherespectivegenesegments.First,Ku70-80andDNA-PKcs
bind to the coding ends and recruit other additional NHEJ factors, includingArtemis,
XRCC4-XLF andDNA ligase IV (Fig. 8a). Next, the Artemis nuclease is activated upon
autophosphorylationofDNA-PKcsandopensthehairpinloopofthecodingends,which
can then be ligated to the other coding ends by the XRCC4-XLF and DNA ligase IV
complex(Lietal.1995;Casellasetal.1998;Maetal.2002).ThecreationofDSBswith
different ends andthe requirement for associationof the12-and23-RS sequences in
human B and T cells is essential for regulated processing to ensure that coding
sequencesofV,DandJ,butnothomotypicgenesegmentsarejoined.Thefinalproduct
ofV(D)JrecombinationisaDNAcodingsequenceconsistingofrandomlyrearrangedV,
DandJgenesegments(SchatzandSwanson2011).
Classswitchrecombination(CSR)andsomatichypermutation(SHM)onlyoccurin
activatedgerminalcenterBcells.CSRleadstothechangeofantibodyisotypeandthus
itseffectorfunction,whileSHMaffectsthevariableregionsoftheimmunoglobulingenes
to promote diversity of antibodies. Both CSR and SHM are independent of RAGs and
insteadaretriggeredbyactivation-inducedcytidinedeaminase(AID)(Muramatsuetal.
2000; Arakawa et al. 2002; Petersen-Mahrt et al. 2002)(Fig. 8b). AID specifically
deaminatescytosinesintouracils,resultingintheformationofU:Gmismatches.During
CSR,theCμexonisexchangedbyCγ/ε/αexons,resultinginthechangeoftheantibody
isotype from IgM to IgG, IgEor IgA (Methot andDiNoia2017).UnlikeRAGs thatare
targeted to very specific sequences, AID in CSR is active on large regions (1-3 kb) of
repetitive DNA sequences upstream of CH genes (coding for constant regions of
immunoglobulinheavy chains), called the switch(S) regions.TheU:Gmismatchesare
mainly processed by the base excision repairmachinery (BER),which creates ssDNA
breaks as intermediates of the repair process (Rada et al. 2002; Imai et al. 2003;
Schrader et al. 2005). Closely spaced ssDNA breaks then give rise to staggered DSBs
(DSBswithssDNAoverhangs)(Fig. 8b).Additionally, a smaller fractionofDNAbreaks
mayformasaresultofanon-canonicalfunctionoftheDNAmismatchrepairmachinery,
which involves the exonuclease activity of Exo1 (Ehrenstein and Neuberger 1999;
25
Schraderetal.2007;Bregenhornetal.2016).TheexonucleolyticprocessingofoneDNA
strandthencollideswithanickintheoppositeDNAstrand, leadingtoDSBs(Fig.8b).
Irrespectively of the exact mechanism, the resulting DSBs often contain ssDNA
overhangs,whichneed tobe either filledor cleavedprior to joiningby the canonical
NHEJmachinery.DSBswithoverhangsbearingmicrohomologiesmayalsobejoinedby
theMMEJpathway(Lee-Theilenetal.2011).AswithV(D)Jrecombination,CSR isalso
dependentonend-joiningmechanisms,andit isthusnotahomologousrecombination
process.Interestingly,MRNwasfoundimportantforCSR,whereitlikelyhasastructural
roletopromotebothcanonicalNHEJandMMEJpathways(Dinkelmannetal.2009).
Somatichypermutationis,asCSR,dependentonAID(Arakawaetal.2002;Phamet
al.2003).IncontrasttoCSR,SHMleadstoantibodydiversificationthroughmutagenesis
intheVregionsoflightandheavychainsofimmunoglobulins.Ithasbeenestimatedthat
themutation rate in theV regionsduringSHMisabout6ordersofmagnitudehigher
thanintherestofthegenome.Themajorityofmutationsaresinglebasesubstitutions,
withasmallfractionofshortinsertionsordeletions.Thissuggeststhattheformationof
a DSB is not an obligate step in SHM, and in fact NHEJ-deficient cells do not show
significant defects in SHM.However, some DSBs form during SHM (Papavasiliou and
Schatz 2000). To this point, it has been demonstrated that DSBs during SHM can be
repairedbyhomologousrecombination(PapavasiliouandSchatz2000;Zanetal.2003);
furthermore, HR has been implicated as a safeguard against off target AID activity
(Hashametal.2010;Zahnetal.2014).Interestingly,MRNwasfoundtopromoteSHM,
buttheunderlyingmechanismsremainunclear(Yabukietal.2005).
Finally,theinsertionofDNAfragmentsofvarioussizeshasbeenrecentlyreported
to occur in the switch region of immunoglobulin genes (Tan et al. 2016; Pieper et al.
2017).TheDNAsequencesoriginatefromanotherpartofthegenomeandthetransfer
occurs via a copy-paste rather than a cut-paste process. Thismay represent a novel
mechanismforantibodydiversification,althoughthemolecularpathwaysrequiredfor
thesetransactionsareunknown.Interestingly,insertionsoffragmentsfromthecollagen
receptorLAIR1intoimmunoglobulingenesleadtoantibodiesthatarebroadlyreactive
tomalaria(Tanetal.2016;Pieperetal.2017).ItisimportanttopointoutthatduringB
cell maturation, clones that produce high affinity antibodies are positively selected
while lowaffinityclonesareeliminated.Due to theselectionprocess,even infrequent
eventsmaythusbecomephysiologicallyrelevant(Pieperetal.2017).
6.2Homologousrecombinationinmeiosis
26
Meiosisisaspecializedcelldivisionthatisrequiredforgenomehaploidization(Keeney
et al. 2014). This is essential to form spores or gametes in sexually reproducing
unicellularormulticellulareukaryotes,andmakessure thatploidy ismaintainedwith
each successive generation (Fig. 9). DSBs are introduced duringmeiosis, and become
substrates for the homologous recombination machinery (Sun et al. 1989; Sun et al.
1991). The function ofmeiotic recombination inmost organisms including yeast and
vertebrates (but notDrosophila and Caenorhabditis) is to make physical connections
betweenhomologouschromosomestofacilitatetheirproperalignmentandsubsequent
segregation.Additionally, recombinationcreatesgeneticdiversity inthepopulationby
exchanging DNA regions between maternal and paternal chromosomes. The key
mechanisticdifferencesbetweenHRinvegetativecellsandduringmeiosisare(a) the
formationofDNAbreaks,whichareinducedinaprogrammedmannerduringmeiosis;
(b)thepreferentialusageofthehomologouschromosomeasatemplateforrepairand
(c)abiasforpreferentialresolutionofrecombinationintermediatesintocrossoversthat
arenon-randomlyspaced(Fig.9)(Hunter2015).
DSBs are introduced during the first meiotic division by the SPO11/Spo11
transesterase,which is evolutionarily conserved fromyeast tomammals (Keeneyand
Kleckner1995;Keeneyetal.1997;Mahadevaiahetal.2001;Robertetal.2016).SPO11
is a topoisomerase-like protein that cleaves both strands of dsDNA, but remains
covalentlyattachedtothe5'-terminatedDNAstranduponcleavage.Inyeast,atleast9
other proteins are required for Spo11 to cleave DNA, including the MRX complex
(Keeney 2008). Interestingly, the nuclease activity of MRE11 is dispensable for DNA
cleavagebySpo11inyeast,butitisinsteadessentialtoinitiatetheprocessingofSpo11-
boundDNAbreaks(Bordeetal.2004;LamandKeeney2014).Aswithotherprotein-
blocked DNA ends, the first resection step requires the nuclease activity of the
MRN/MRX complex with its co-factor CtIP/Sae2 to remove SPO11/Spo11 from the
breakends(KeeneyandKleckner1995;Nealeetal.2005).ResectionbyMRX-Sae2can
proceeduptoseveralhundredofnucleotidesinlength.Thisisfollowedbylong-range
resection, which appears to be exclusively carried out by the Exo1 branch in yeast
meiotic cells (Zakharyevich et al. 2010; Mimitou et al. 2017). In mice, the average
resectionlengthis0.9kb(Langeetal.2016),whichissimilartothevalueobtainedin
yeast(Zakharyevichetal.2010).
The resected3’DNA tail is boundbyRPAand successively replacedbyDMC1(a
meiosis specific strand exchange protein) together with RAD51 (Bishop et al. 1992;
Cloud et al. 2012).Whereas RAD51 is the only strand exchangeprotein in vegetative
cells, meiotic cells employ both RAD51 and DMC1. In addition to the recombination
27
mediatorssuchasyeastRad52andhumanBRCA2thatfunctionduringrecombinationin
bothvegetativecellsand inmeiosis,meioticcellsmakeuseofanadditionalregulator,
the HOP2-MND1 complex (Leu et al. 1998; Petukhova et al. 2005). The heterodimer
stabilizestheRAD51/DMC1complexonssDNA,reducestheaffinityofRAD51todsDNA
andmodulatestheconformationofthenucleoproteinfilamenttopromoteDNAstrand
exchange(Chietal.2007;Pezzaetal.2007).
ThesecondkeydissimilaritybetweenmeioticandmitoticHRisthetemplatechoice
forrepair.Meioticcellspreferentiallyuse thehomologouschromosome insteadof the
sister chromatid in order to fulfill the requirement to generate genetic variability
(Schwacha and Kleckner 1994; Baudat et al. 2000; Peoples et al. 2002). How this is
achievedisnotyetfullyunderstood.Thetemplatebiasisregulatedbycohesion(Kimet
al. 2010), depends inpart on the role of DMC1 to promote strand exchange between
homologs, and requires the DNA damage response cascade involvingMec1, Tel1 and
meiosis-specific components such as Mek1 (Schwacha and Kleckner 1997; Niu et al.
2005;Carballoetal.2008).
The third key characteristic of meiotic HR is a more frequent resolution of joint
molecule intermediatessuchasdHJsor theirprecursors intocrossovers.Mostmeiotic
non-crossover products appear early and result from the processing of unstable
intermediates by SDSA (Allers andLichten2001;Bishop and Zickler 2004). The joint
molecules that can be readily observed are termed single-end invasions (containing
presumably D-loop structures), which later mature into dHJs (Hunter and Kleckner
2001). Most of these joint molecules are resolved synchronously later, which is
triggeredbythephosphorylationofyetundefinedtargetsbytheCdc5kinase(Allersand
Lichten 2001; Sourirajan and Lichten 2008). Although the molecular mechanism
remains unclear, the preferential processing of joint molecule intermediates into
crossoversisachievedbytheusageofameiosis-specificpro-crossoverpathway,which
isresponsibleforupto~80%ofmeioticcrossoversinS.cerevisiae(Zakharyevichetal.
2012). This pathway ensures that every chromosome receives at least one crossover
(positivecrossoverinterference),therebyassuringtheexchangeofgeneticinformation
between the homologous chromosomes. At the same time, the formation of one
crossover suppresses the formation of additional crossovers in its vicinity (negative
crossover interference), making sure that the crossovers are evenly spaced, as two
crossoversinimmediatevicinitylimittheexchangeofgeneticinformationbetweenthe
homologouschromosomes.Thepropernumberof crossoversandtheirdistribution is
thus balanced to enable efficient chromosome segregation and genetic exchange
(Hunter2015).
28
Theexactmechanismofcrossoverinterferenceremainstobedefined.Itisapparent
that it is regulated by the components of the synaptonemal complex that juxtaposes
homologs along their entire length (Sym and Roeder 1994; Hunter 2015). The
interference signaling involves Tel1 (Garcia et al. 2015). Crossover interference also
requirestopoisomeraseII,suggestingthatinterferencemayberegulatedbymechanical
or topological stress along the meiotic chromosome axes (Zhang et al. 2014). The
meioticcrossover-specificpathwayinvolvestheZMMgroupofproteins,whichincludes
Zip1, Zip2, Zip3, Zip4, Spo16,Mer3andMsh4-Msh5 inS.cerevisiae (Lynnet al. 2007;
Shinohara et al. 2008). These proteins form or facilitate the formation of the
synaptonemalcomplex,orfunctionmoredownstreamtostabilizeD-loopstructuresina
waythatprotectstheirdisassemblybymotorproteins(Mazinaetal.2004;Snowdenet
al.2004).Similarlyinmice,theputativeSUMO-conjugatingfactorRNF212wasfoundto
stabilize meiotic recombination factors including MSH4-MSH5 at designated
recombination sites, which is essential for crossing over. This is likely achieved via
sumoylationofselectedtargetsalongthechromosomeaxes,whichfunctionallyinteracts
with HEI10-dependent ubiquitination and proteasomal degradation (Wei et al. 2003;
Reynoldsetal.2013;Raoetal.2017).
Thecrossover-specificresolution inbuddingyeast,plantsandmammalsdepends
onthenucleaseactivityofMlh3,whichisapartoftheMlh1-Mlh3heterodimer(Lipkinet
al.2002;Nishantetal.2008;Zakharyevichetal.2012;Ranjhaetal.2014;Rogachevaet
al.2014).Unexpectedly,Exo1was foundtohaveanon-catalytic function incrossover
resolutiontogetherwithMlh1-Mlh3(Zakharyevichetal.2010).HowtheZMMproteins
and Mlh1-Mlh3 achieve the crossover-specific resolution of dHJs or their precursors
remains to be described (Fig. 7c). Interestingly, only crossovers from the ZMM-Mlh1-
Mlh3pathway– termedClass Icrossovers–display interference. Jointmolecules that
areprocessedbythestructure-specificnucleases(Mus81-Mms4,Slx1-Slx4andYen1in
yeast) result in both crossovers and non-crossovers. Crossovers resulting from these
nucleases are interference independent and account for ~20% of crossovers in S.
cerevisiae(delosSantosetal.2003;Zakharyevichetal.2012).Incontrast,Mus81-Eme1
generates most meiotic crossovers in the budding yeast Schizosaccharomyces pombe,
while the MEI-9, an XPF-like protein, resolves meiotic joint molecules in Drosophila
(Boddyetal.2001;Yildizetal.2002).
7 Role of recombination proteins in promoting the stability of DNA replication
forks
29
In addition to repairingDSBs, recombinationproteins have critical functions in other
pathwaysofDNAmetabolism. Specifically, recombination is oneof thepathways that
contribute to the repair of DNA crosslinks, which has been covered elsewhere (Hinz
2010). Furthermore, recombination in multiple ways promotes the stability of
replicating DNA (Branzei and Szakal 2017). Abnormalities such as protein blocks,
chemicalmodifications,abnormalsecondarystructuresorcollisionsofreplicationforks
withthetranscriptionmachinerycanimpedeDNAreplication.Dependingonthenature
ofthereplicationstress,cellsutilizevariousmechanismsthathelpthemdealwiththese
situations(Fig.10a-d).Thisincludesre-primingofDNAreplicationand/ortranslesion
DNAsynthesis,whichleadtolesionbypass.Postreplicativerepaircanfillanygapsleft
behind by the replication machinery (Bianchi et al. 2013; Garcia-Gomez et al. 2013;
Mouronetal.2013;BranzeiandSzakal2016;GuilliamandDoherty2017;Vaismanand
Woodgate2017).Insomecasesthereplicationforkcanfallapart,resultinginasingle-
endedDSB,which is repairedasdescribed in chapter1.3.Here,wewill focuson two
related functionsofrecombinationproteins thatwereuncoveredonly inrecentyears,
namely in the replication fork reversal and the protection of DNA from nucleolytic
degradationatstalledreplicationforks.
ReplicationforkreversalistheconversionofastalledDNAreplicationforkintoa
four-way junction (Fig. 10b). Fork reversal is achieved by the annealing of the two
nascentDNAstrandsandthere-annealingofthetwoparentalstrands,whichresembles
aDNAdouble-strandbreak.ForkreversalcanthusleadtoDSBformationintheabsence
of templatebreakage(Fig.10b).Althougharisingthroughaverydifferentmechanism,
theresultingfour-wayjunctionisstructurallyidenticaltoaHollidayjunctionthatforms
duringDNArecombination(Fig.10b).Theconceptofreplicationforkreversalhasbeen
proposedalongtimeago(Higginsetal.1976).Inbacteria,ithasbeenestablishedasa
waythatcontributesto therestartofstalledreplication forks. InE.coli, theactionsof
RecA(ahomologueofRAD51), followedbybranchmigrationbyRuvAB,or themotor
activityofRecG,candrivereplicationforkreversal(Seigneuretal.1998;McGlynnand
Lloyd2001;Guptaetal.2014).Foralongtime,thisprocesswasbelievedtorepresent
only a pathological reaction in eukaryotic cells (Sogo et al. 2002; Hu et al. 2012).
Specifically, forkreversal inyeastwasdescribed tooccur incheckpoint-defectivecells
(Lopes et al. 2001). Only recently, it has been demonstrated that replication fork
reversalcanbebeneficialtopreventDNAtemplatebreakageinhumancells,andthatit
isaglobalresponsetoDNAreplicationstress(RayChaudhurietal.2012;Neelsenand
Lopes 2015; Zellweger et al. 2015). Electronmicroscopy revealed that the regressed
30
arms of the replication forks can reach and occasionally exceed 1 kb in length,
implicatingthattheyforminthecourseofanactiveandenzyme-drivenprocess.
One of the first proteins shown to promote replication fork reversal in vivo was
RAD51(Zellwegeretal.2015).Inaccord,RAD51,toalimitedextent,candrivemigration
ofHolliday junctions viapolymerizationonDNA (Rossi et al. 2011).However,RAD51
persehasnomotoractivity,soit isunlikelythatRAD51candriveforkreversalonits
own.ThebestcandidatesforthisfunctionaremotorproteinssuchasDNAhelicasesor
translocases. To this point, the motor protein RAD54 was shown to catalyze fork
reversal invitro in away thatwas stimulatedbyRAD51 (Bugreev et al. 2011).Other
proteins, including SMARCAL1, ZRANB3, HLTF, FBH1, RECQ5 and FANCM were
proposedtofunctioninforkreversalinvitroorinvivo(Kanagarajetal.2006;Garietal.
2008;Betousetal.2012;Cicciaetal.2012;Bhatetal.2015;Fuggeretal.2015;Kileetal.
2015;Kolinjivadietal.2017;Taglialatelaetal.2017;Vujanovicetal.2017).However,
the interplay of these factorswith RAD51 isnotdefined, and it is not clear towhich
extent these motor proteins can function in a redundant manner. Interestingly, fork
reversallikelydoesnotrequireBRCA2,showingthat,unlikeinDSBrepair,thefunction
of RAD51 in promoting fork reversal is BRCA2 independent (Prakash et al. 2015;
Kolinjivadietal.2017;Mijicetal.2017).ForkreversalallowstimeforDNArepairahead
of the fork,ormayresult inDNAdamagebypass(i.e.damage tolerance, incasewhen
onereversedarmtemplatesDNAsynthesisofthecomplementarystrand).Oncethisis
completed,DNAreplicationmustresume.Severalmechanismshavebeendescribedthat
promote the restoration of reversed replication forks, which involve motor proteins
suchasRECQ1,RAD54orthenucleolyticdegradationofthereversedarmsbyMRE11,
EXO1,theDNA2-WRNnuclease-helicasepairorbytheMUS81-dependentcleavage,with
different implication forgenomestability (Bugreevetal.2011;Bertietal.2013;Pepe
andWest 2014a; Thangavel et al. 2015; Lemaconet al. 2017).While replication fork
reversalmaybepathologic insomecases, ithasbeendemonstrated that inhibitionof
forkreversalresults in increasedDNAbreakageandgenome instability,underpinning
theroleofthisprocessinpreventinggenomerearrangements(NeelsenandLopes2015;
Mijicetal.2017).
As described in chapter 3, RAD51 is the main DNA strand exchange protein in
eukaryotes. It remains to be defined whether RAD51 has a structural or a catalytic
functioninreplicationforkreversal.Thereis,however,onekeyfunctionofRAD51that
isDNA repairand recombination independent. Ithasbeendemonstrated thatBRCA1,
BRCA2andRAD51 functiontogether toprotectstalled–andpossiblyreversed–DNA
replication forks from degradation by the MRE11 nuclease (Hashimoto et al. 2010;
31
Schlacheretal.2011;Schlacheretal.2012;Mijicetal.2017).Ithasbeenshownthata
stable RAD51 nucleoprotein filament is required to protect DNA from MRE11
(Kolinjivadietal.2017;Zadorozhnyetal.2017).TheBRCA1andBRCA2 factors likely
act directly or indirectly to stabilize the RAD51 nucleoprotein filament. It is being
discussedwhether the essential function of BRCA2 is to prevent DNA degradation at
stalledforksbystabilizingRAD51,ortopromotecanonicalDSBrepair(RayChaudhuri
et al. 2016; Feng and Jasin 2017). At the same time, unrestricted RAD51 activity at
stalled forks is equally detrimental: the RADX factor was recently identified as a
negative regulator of RAD51 at stalled forks (Dungrawala et al. 2017). These
experiments collectivelydemonstrated that inDNA replication, theRAD51protein, in
addition to performing canonical "recombination" functions has other, previously
unrecognized roles in replication fork reversal and protection from nucleolytic
degradation. To elucidate the underlying molecular mechanisms of these processes
representsachallengetoresearchersinthecomingyears.
Figurelegends
Fig.1AnoverviewofDNAbreaks.aAsingle-strandedDNA(ssDNA)breakariseswhen
only one strand of double-stranded DNA is interrupted. b If an ssDNA break is
encountered by DNA replication, it gives rise to a one-ended double-stranded DNA
(dsDNA) break. c A two-ended dsDNA break forms when dsDNA is broken into two
pieces.
Fig. 2 Anoverviewof the twomainpathways forDNAdouble-strandbreak repair in
human cells.a Main differences between end-joining and homologous recombination
pathways. b DNA double-strand break repair pathway usage gives rise to different
outcomesduringgenomeeditingwithCRISPR-Cas9.Whereasend-joiningoftenresults
inrandommutationsinthevicinityofthebreaksitethatmaydisruptthereadingframe
ofthetargetedgene,homologousrecombinationmaymediatetheprecisereplacement
ofgeneticinformation.
Fig.3AnoverviewofDNAend-joiningrepairmechanisms.aOverviewandmainfactors
of non-homologous end-joining. b Overview and main factors of microhomology-
mediatedend-joining.
32
Fig.4Anoverviewofhomologousrecombinationpathways.Aschematicrepresentation
ofa single-strandannealing,b synthesis-dependentstrandannealing,cbreak-induced
replication and d canonical DNA double-strand break repair pathway that involves
generation of a doubleHolliday junction,which can be processed by either topologic
dissolution(d1)ornucleolyticresolution(d2).Thevariouspathwaysdifferintermsof
mutagenicpotentialandwhethertheyleadtocrossoverornon-crossoverproducts,as
indicated.ThegreentrianglesindicateDNAreplicationsites.NewlysynthesizedDNAis
illustratedusingdashedlines.
Fig. 5 An overview of DNA end resection in human cells. The first (short-range)
resection step involves theMRE11 nuclease and the second (long-range) step either
EXO1orDNA2nuclease.DNA end resection leads to the generationof 3' overhanged
DNAatDNAdouble-strandbreaksites.ThedashedbluelinesindicatedegradedDNA.
Fig. 6 An overview of the RAD51 filament formation (presynaptic phase) and the
invasion of template dsDNA (postsynaptic phase) in human cells. Both positive and
negativeregulatorsoftheprocessareindicated.
Fig.7AnoverviewofdoubleHollidayjunctionprocessingmechanismsinthecanonical
recombinational DSBR pathway. In vegetative cells, double Holliday junctions can be
processedbyeitherdissolution(a)thatinvolveshelicase-coupledtopoisomeraseorby
resolution(b) that involvesnucleolyticcleavageof the junctions.Meioticcells inmost
organismspreferentiallyuseadedicatedcrossoverpathway(c),andtoasmallerdegree
nucleasesthatarecommontobothmeioticandmitoticcells(b).Forsimplicity,human
nomenclature isused.The involvementofnucleases listed inpanelsbandcinhuman
(ormouse)meioticcellsgenerallyremainstobedefined.YeastS.cerevisiaecellsusea
crossover-specific pathway that involves the Exo1-Mlh1-Mlh3 complex and the
structure-specificnucleasesMus81-Mms4,Slx1-Slx4andYen1.Seetextfordetails.
Fig. 8 An overview of specialized roles of the DSB repair pathways in human
lymphocytes.aMechanismofV(D)Jrecombination,whereDNAbreaksare introduced
by RAG1-RAG2. b Mechanism of class switch recombination, where DNA breaks are
indirectly causedby the actionofAID. Inboth cases end-joining, butnothomologous
recombinationpathways,areresponsibleforDNAbreakrepair.
33
Fig. 9 Anoverviewof specialized rolesof thehomologous recombinationpathway in
meiosis. Themaindifferencesbetween recombination inmeiotic andvegetative cells
areindicated.
Fig. 10Anoverviewofpathways thatrelieveDNAreplicationstress inhumancells.a
WhenaDNAreplicationforkencountersanobstacle,itmaybebypassedbytranslesion
synthesis.b A DNA replication fork can also reverse, whichmay temporarily protect
DNAfrombreaking.Replicationforkreversalcanalsoleadtolesionbypass.Factorsthat
may regulate the various steps of replication fork reversal and restart are indicated.
RAD51likelymediatesreplicationforkreversalandprotectsreversedreplicationforks
from degradation by the MRE11 nuclease. c Replication repriming downstream of a
lesion is another mechanism of DNA damage tolerance. d Replication forks can also
break,andbecomeasubstrateforhomologousrecombinationasindicatedinFig.4.
Acknowledgements:Wewould like to thankmembers of the Cejka laboratory (IRB
Bellinzona), Massimo Lopes (University of Zurich) and Kathrin Pieper (Lanzavecchia
laboratory,IRBBellinzona)forcommentsonthemanuscript.Theworkwassupported
bygrantsfromtheEuropeanResearchCouncil(681630)andtheSwissNationalScience
Foundation(31003A_175444)toP.C.Weapologizetocolleagueswhoseworkcouldnot
becitedduetolengthconstraint.
The authors declare no conflict of interest. This article does not contain studies
involvinganimalsorhumanparticipantsbyanyoftheauthors.
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ssDNA breakreplisome
parental stranddaughter strand
parental strand
brokendaughter strand
a
b
cssDNA break two-ended
dsDNA break
one-endeddsDNA break
Figure 1
dsDNA break
mutations
DNA template
• Fast • Slow• Template independent • Requires template• Often mutagenic • Largely accurate• Cell cycle independent • Only S/G2 phase• Only two-ended breaks • Both one and two-ended breaks• No repair of protein blocked ends • OK for protein blocked ends
Cas9
STOP
DNA break repair
End-joining Homologous recombination (HR)
DNA template
Random mutagenesis, frameshiftleads to premature termination
Precise editing, replacement with desired DNA sequence
a
b
End-joining: Non-homologousend-joining (NHEJ) or Microhomologymediated end-joining (MMEJ)
Homologous recombination (HR)
Figure 2
mutations
guide RNA
target dsDNA
Overview of end-joining DNA double-strand break repair pathways
Non-homologousend-joining (NHEJ)
Microhomology-mediatedend-joining (MMEJ)
microhomology at ends (2-20 nt)
two-endeddsDNA break
Dna2, MRN, CtIP, ???
Dna2, MRN, CtIP, ???
Polθ
DNA Ligase III, ???
DNA overhangcleavage
DNA synthesis DNA synthesis
DNA ligation DNA ligation
DNA end protection
limited DNA end processing
no or short (< 4 nt) microhomology at ends
DNA annealing
Ku70-Ku80
XRCC4-XLF-DNA Ligase IV
ArtemisPolλ or Polμ
DNA-PKcsin case of nonligatable DNA ends:
DNA-PKcs
Figure 3
a b
oror
or
dsDNA break
Extended DNA end resection
Annealing at homologous(>20 nt) loci, overhang cleavage
DNA synthesis
Endpoint: non-crossover,deletion at break site
Single-strand annealing (SSA)
Synthesis-dependent strand annealing (SDSA)
Break-induced replication (BIR)
Topological processing of dHJ (Dissolution)
Nucleolytic cleavage of dHJ (Resolution)
Overview of homologous recombination pathways
DNA synthesis
Endpoint: non-crossover
Disruption of D-loop
Invasion of homologous DNA
Second end captureDNA synthesis
DNA synthesis, ligation
D-loop
3’ ssDNA overhang
double Holliday junction (dHJ)
Migration of bothjunctions towardseach other
Endpoint: non-crossover
Endpoint: non-reciprocalcrossover
DNA synthesis viabubble migration
... Later in cell cycle
Cleavage by structure-selectivenucleases along horizontal orvertical lines (random)
Endpoint: reciprocal crossover
Endpoint: non-crossover
Cano
nica
l DN
A d
oubl
e-st
rand
bre
ak re
pair
path
way
via
dou
ble
Hol
liday
junc
tion
template DNA
a
b
c
d1
d2
Figure 4
dsDNA breakOverview of DNA end resection pathways
short 3’ ssDNA overhang
DNA adduct
MRE11RAD50
NBS1
Recruitment of MRN complex
CtIP
Recruitment of phosphorylated CtIP
P
Activation of MRE11endonuclease
EXO1WRN or BLMDNA2
5’3’
3’3’
or
Shor
t ran
ge re
sect
ion
long 3’ ssDNA overhanglong 3’ ssDNA overhang
5’
Resection backtoward endMRE11 exonuclease
1st step:Short-range DNA end resection
2nd step:Long-range DNA end resection
Figure 5
RPA
RAD51BRCA2
RAD51 �lament
remodelled RAD51 �lament
D-looptemplate DNA
DNA synthesis
RAD54
Resected RPA-coated3’ ssDNA overhang
3’
BRCA2 exchanges RPA forRAD51 on overhanged ssDNA,blocks dsDNA binding of RAD51
RAD51 nucleprotein �lament
Active RAD51 �lament
Invasion of template dsDNA, RPA stabilizes D-loop by binding to displaced ssDNA
Removal of RAD51 from dsDNA by RAD54, DNA synthesis by Polδ or Polε
RAD51 paralogsRAD54RAD52PALB2
RAD51 paralogs
BRCA1
RAD51 paralogsDSS1SWS1 ?
RECQ5FBH1PARIBLM
RTEL1BLMRECQ1RAD54
RPA
Pres
ynap
tic
phas
ePo
stsy
napt
ic p
hase
Positiveregulation:
Negativeregulation:
RAD54
Figure 6
Convergent branch migration (dissolution)
Nucleolytic resolution
double Holliday junction (dHJ)
Hemicatenane
Endpoint: non-crossover
Convergently migrated dHJ
Nucleolytic resolution interference-dependent
Endpoint: Crossover
Endpoint: non-crossover
OR
Endpoint: Crossover
BLM-TopoIIIα-RMI1-RMI2
SLX1-SLX4 MUS81-EME1(XPF-ERCC1) SM
XSM
GEN1
MLH1-MLH3 and EXO1, ZMM proteins, ?
Mechanism:Helicase & topoisomerase
Helicase & topoisomerase
Topoisomerase
Mechanism: Nuclease
Mechanism: Nuclease
Vegetative cells Meiotic cells
a
b
c
Figure 7
Recognition of RS12/23 by RAG1-RAG2
NHEJ-mediated DSB repair
Vn Vn + 1 Jn Jn + 1 Jn + 2 Jn + 3
DNA cleavage by RAG1-RAG2
Lost
RS23 sequenceRS12 sequence
hairpin endblunt end
properly joined V and J segments
MRNArtemis
Ku70-Ku80DNA-PKcs
XRCC4-XLF, DNA ligase IV
circular DNA
V(D)J Cμ Cδ Cγ Cε
V(D)J Cμ Cδ Cγ Cε
UG
GU
GG
G
G
GG
G
G
IgM
V(D)J
Cμ Cδ
Cγ Cε
EXO1
Mismatch repair
Base excision repair
DNA double-strand break
+
+
Lost
V(D)J Cγ Cε
IgG
NHEJ or MMEJ-mediated DSB repair
RAG1-RAG2RAG1-RAG2
Vn Vn + 1 Jn Jn + 1 Jn + 2 Jn + 3
Loop formation, alignment of RS12 and RS23 sequences
a V(D)J recombination b Class switch recombination
MRN
AID
T cellsB cells
AID
Figure 8
Cα
Cα
Cα
Cα
Diploid cell
Haploid gametes
dsDNA break formation
Homologous recombination
Crossover formation
1 meioticdivision
2 meioticdivision
Meiotic vs. Vegetative cells
DNA double-strand breaks:SPO11-induced, programmed Accidental
Preferential repair template:Homologous chromosome Sister chromatid
Genetic outcome:At least one crossover perchromosome, evenly spaced
Non-crossovers >> crossovers
Maintenance of genome stability Proper chromosome seggregationExchange of genetic information
st
nd
Strand exchange protein(s):RAD51, DMC1 RAD51
FUNCTION:
Figure 9
replisome
parental strand
lagging strand
STOPlesion
leading strand
parental strand
lagging strand
STOP
lesion
leading strand
RAD51, RAD54 SMARCAL1ZRANB3FBH1, RECQ5FANCM, ?
RAD51 �lament
MRE11PTIP?
replisome
parental strand
lagging strand
leading strandRECQ1
MUS81-EME1/EME2RAD54RAD51 ?
replisome
parental strand
lagging strand
leading strand
Swith back to replicative polymerase
translesion polymerase
Postreplicative gap repair
Lesion ahead of replication fork
ContinuedDNA replication
RADX
BRCA2
a Translesion synthesis
b Replicationfork reversal
c Replicationrepriming
d Break inducedreplication
Replication fork restoration
Fork breakageMUS81-EME1RECQ5
See Figure 4
lesion
lesion
Figure 10
STOP
STOP