Download - The endonuclease IV family of apurinic/apyrimidinic endonucleases

Mutation Research 705 (2010) 217–227

Mini-review

The endonuclease IV family of apurinic/apyrimidinic endonucleases

James M. Daley, Chadi Zakaria, Dindial Ramotar *

Centre de Recherche, Hopital Maisonneuve-Rosemont, Universite de Montreal, 5415 de L’Assomption, Montreal, QC H1T 2M4, Canada

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

2. Sources and biological consequences of AP sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

3. Repair of AP sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

4. E. coli endonuclease IV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

5. S. cerevisiae Apn1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

5.1. The role of Apn1 in mitochondrial BER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

6. S. pombe Apn1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

7. C. elegans APN-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

8. Conclusions and future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

A R T I C L E I N F O

Article history:

Received 27 April 2010

Received in revised form 3 July 2010

Accepted 14 July 2010

Available online 3 August 2010

Keywords:

Endonucleases

Base excision repair

Nucleotide incision repair

Genomic stability

Yeast

C. elegans

A B S T R A C T

Apurinic/apyrimidinic (AP) endonucleases are versatile DNA repair enzymes that possess a variety of

nucleolytic activities, including endonuclease activity at AP sites, 30 phosphodiesterase activity that can

remove a variety of ligation-blocking lesions from the 30 end of DNA, endonuclease activity on oxidative

DNA lesions, and 30 to 50 exonuclease activity. There are two families of AP endonucleases, named for the

bacterial counterparts endonuclease IV (EndoIV) and exonuclease III (ExoIII). While ExoIII family

members are present in all kingdoms of life, EndoIV members exist in lower organisms but are curiously

absent in plants, mammals and some other vertebrates. Here, we review recent research on these

enzymes, focusing primarily on the EndoIV family. We address the role(s) of EndoIV members in DNA

repair and discuss recent findings from each model organism in which the enzymes have been studied to

date.

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1. Introduction

Genomic DNA is constantly damaged by a variety of endoge-nous and exogenous agents. Base damage is one of the mostcommon forms of DNA damage. Oxidation, alkylation anddeamination of bases can disrupt base pairing and promotemutagenesis by leading to misincorporation errors during DNAreplication. It is therefore critical for cells to recognize andfaithfully repair these lesions to avoid cancer and premature aging.All kingdoms of life possess apurinic/apyrimidinic (AP) endonu-cleases, which participate in processing base lesions at various

* Corresponding author. Tel.: +1 514 252 3400x4684; fax: +1 514 252 3430.

E-mail address: [email protected] (D. Ramotar).

1383-5742/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.mrrev.2010.07.003

steps. AP endonucleases are named for their ability to cleave APsites in DNA, which are formed when DNA glycosylases cleavedamaged bases at the N-glycosidic bond. Some AP endonucleasesare also endowed with the ability to directly incise the DNA strandupstream of the primary lesion in a process termed nucleotideincision repair (NIR), leaving the damaged base on a 50 flap [1].Finally, AP endonucleases have 30 phosphodiesterase activity,which can process damaged 30 ends on single-strand breaks, and30–50 exonuclease activity which can remove nucleotides from theend of a nick [2,3].

The two families of AP endonucleases are named for theirarchetypal Escherichia coli members, endonuclease IV (EndoIV) andexonuclease III (ExoIII). The two families are structurally unrelated,indicating that they evolved independently and not from acommon ancestor. Table 1 summarizes the key model organisms

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Table 1Summary of organisms in which EndoIV homologs have been identified and their

enzymatic activities.

Organism Identity

to E. coli

EndoIV

Activities

Saccharomyces cerevisiae Apn1 42% AP endonuclease

30 exonuclease

30 phosphodiesterase

NIR endonuclease

Schizosaccharomyces pombe Apn1 42% Uncharacterized

Caenorhabditis elegans APN-1 47% AP endonuclease

30 exonuclease

30 phosphodiesterase

NIR endonuclease

Danio rerio APN-1 48% Uncharacterized

Xenopus tropicalis

APN-1-like protein

45% Uncharacterized

J.M. Daley et al. / Mutation Research 705 (2010) 217–227218

in which EndoIV homologs have been identified and studied. InSaccharomyces cerevisiae, the EndoIV homolog Apn1 is responsiblefor the vast majority of AP endonuclease activity in the cell [2,4]. AnEndoIV homolog (APN-1) also significantly contributes to DNArepair in Caenorhabditis elegans [5]. In Schizosaccharomyces pombe,an EndoIV homolog exists but seems to play only a backup role inDNA repair [6]. EndoIV family members are also present in somefish (Danio rerio) and frogs (Xenopus tropicalis) but have not yetbeen studied in these organisms. EndoIV homologs are curiouslyabsent from higher organisms including insects and mammals.These organisms instead depend solely on ExoIII family members,such as human APE1, for AP endonuclease activity. Unlike EndoIV,ExoIII family members exist in all kingdoms of life. Like humancells, E. coli utilizes ExoIII as its major AP endonuclease, but alsoinduces EndoIV under stress conditions [7–9]. In contrast, the yeastExoIII homolog, Apn2, constitutes only a minor portion (<3%) of APendonuclease activity in extracts and is thought to act as a backupmechanism [2,4,10]. Thus, the currently available data suggest agreater reliance on ExoIII-like AP endonucleases in higherorganisms, whereas EndoIV family members are more importantin simpler organisms. It is unclear which enzyme is evolutionarilyolder, as members of both families can be found in several speciesof archaea that have been sequenced to date. A key differencebetween the two families is that EndoIV members utilize threeZn2+ ions and a hydroxide to cleave the phosphodiester bond,whereas ExoIII members require Mg2+ [11–14]. Thus, sensitivity toEDTA can be used to discriminate between the activities of the twoenzymes [15]. Here, we review recent findings on the biologicalroles of the EndoIV family members in each of the organisms inwhich they have been characterized, with an emphasis on how themultiple activities of these enzymes are integrated into the broaderpicture of DNA repair.

2. Sources and biological consequences of AP sites

Although the AP endonucleases cleave a variety of substrates,they show the greatest in vitro activity on the AP sites for whichthey are named, suggesting that AP sites are their primary target in

vivo [16]. AP sites are among the most common and toxic non-helixdistorting DNA lesions and can be formed spontaneously via slowhydrolysis of the N-glycosidic bond that links the base to the DNAbackbone [17]. Purines are much more susceptible to spontaneousloss than pyrimidines [17,18]. Endogenous AP sites are alsoproduced by the enzymatic action of DNA N-glycosylases thatremove damaged or mispaired bases [19]. In yeast, the observationthat deletion of the uracil glycosylase UNG1 rescues lethality inapn1 apn2 rad1 cells, which lack the key activities to process AP

sites, suggests that uracil misincorporation into DNA is a majorsource of endogenous AP sites [20]. Various modified oxidized APsites are also generated by free radical attack on bases anddeoxyribose sugars. Certain exogenous chemical agents, such asthe alkylating agent methyl methane sulfonate (MMS), theantitumor drug bleomycin, and near-ultraviolet light also contrib-ute to the formation of AP sites, typically by inducing base damagewhich is then converted to AP sites by DNA glycosylases [21].

AP sites have long been known to be mutagenic. Mutationscaused by AP sites were first observed by analysis of single-stranded bacteriophage DNA passaged through E. coli [22,23]. Therecovered DNA contained mainly A:T to T:A transversion muta-tions, suggesting a preference for the incorporation of dAMPopposite the AP site during DNA replication. Indeed, AP sitesreplicated in vitro by a variety of bacterial DNA polymerases alsoshowed this dAMP preference [24–28]. Based on these findings, the‘‘A-rule’’ was proposed, stating that DNA polymerases preferen-tially catalyze non-instructional insertion of adenine opposite anAP site [29]. Consistent with this notion, E. coli mutants defective inrepairing AP sites also showed higher levels of A:T to T:Atransversions [30]. The A-rule does not seem to apply toeukaryotes, however. Studies in S. cerevisiae have uncovereddifferent nucleotide preferences depending on the assay used[31–36]. Despite differences in the pattern of mutations inducedby AP sites across species, it is clear that unrepaired AP sitesare mutagenic and must be repaired rapidly to avoid genomeinstability.

In vitro experiments have shown that AP sites strongly blockreplicating DNA polymerases, leading to stalled replication forksin vivo [36–39]. DNA synthesis can be resumed via error-free post-replication repair, in which recombination-dependent replicationfork reversal allows the polymerase to continue past the lesion[reviewed in [40]]. Alternatively, error-prone translesion synthe-sis can bypass the AP site [reviewed in [41]]. In vitro studies withyeast polymerases have shown that Pol d, Pol h, and Rev1 arecapable of inefficiently inserting a nucleotide across from an APsite, although each polymerase preferentially inserts a differentnucleotide [36,42]. These polymerases then stall because they areunable to extend the inserted nucleotide paired with the AP site[36,42]. Pol z cannot insert a nucleotide across from the AP site,but can extend the nucleotide inserted by the other polymerases,leading to a two-polymerase model for AP site bypass [36,43,44].The ability of several polymerases with different preferences toinsert a nucleotide across from an AP site explains why yeast doesnot follow the A-rule and provides mechanistic insight into thepattern of mutations observed in this organism. Genetic evidencealso suggests that AP sites can block transcribing RNA poly-merases and that this process increases mutagenesis, but themechanism by which a stalled RNA polymerase might lead tomutations is currently unknown [45]. Besides blocking poly-merases, AP sites can cause topoisomerase I to become trapped onDNA [46].

3. Repair of AP sites

AP sites are repaired primarily by base excision repair (BER).This pathway is initiated when an AP endonuclease cleaves thesugar-phosphate bond 50 of the AP site to produce a single-strandbreak with 30 hydroxyl (OH) and 50 deoxyribose phosphate (dRP)termini (Fig. 1, Fig. 2A). At this point, repair can be completed byeither short-patch (SP) or long-patch (LP) BER [reviewed in [47]]. InSP-BER, the 50 dRP is removed by a 50 dRP lyase to create a singlenucleotide gap, which is resynthesized by a DNA polymerase(Fig. 1). The nick is sealed by a DNA ligase to complete repair. In LP-BER, displacing synthesis of�2–14 nucleotides forms a 50 flap withthe dRP on the end (Fig. 1). The flap is then cleaved by flap

[(Fig._1)TD$FIG]

Fig. 1. General model for AP endonuclease involvement in base excision repair and related pathways. Damaged bases can be incised to initiate nucleotide incision repair (left)

or removed by glycosylases to form AP sites (right). The damaged base is resynthesized by either short- or long-patch repair. Strand breaks with damaged termini (far right)

are also repaired by the AP endonucleases.

J.M. Daley et al. / Mutation Research 705 (2010) 217–227 219

endonuclease 1 (FEN-1) and, as in SP-BER, a DNA ligase seals thenick.

AP sites can also be cleaved by AP lyases [48]. E. coli

endonuclease III (EndoIII) is the prototypical AP lyase, and manyDNA glycosylases in higher organisms have additional AP lyaseactivity [49–51]. Unlike AP endonucleases, AP lyases cleave on the30 side of the AP site through a b-elimination reaction to producean a,b-unsaturated aldehyde [52–54]. This product blocks DNArepair synthesis and must therefore be removed by a 30

phosphodiesterase before repair can proceed [11]. Most APendonucleases possess this activity and are capable of removinga variety of 30 blocking groups in addition to a,b-unsaturatedaldehydes, including 30 phosphates and 30 phosphoglycolates,which are present at single-strand breaks caused by oxidizingagents (Fig. 2B) [3,15,55]. Genetic data in E. coli and S. cerevisiae

suggests that AP sites are mainly processed by the AP endonu-cleases, with the AP lyases serving as a backup mechanism [50,56–58]. The situation appears to be reversed in other organisms suchas S. pombe, however, where the lyase pathway predominates[59,60].

4. E. coli endonuclease IV

The first indication regarding the presence of a specific class ofenzymes capable of processing AP sites came from studies in E. coli.ExoIII was the first AP endonuclease discovered and accounts for�90% of the total AP endonuclease activity in the cell [61]. ExoIIIwas first described as a 30 to 50 exonuclease, and it was originallythought that the major cellular AP endonuclease activity was dueto a separate enzyme termed endonuclease II [62,63]. Later, it wasfound that the same mutation disrupts both exonuclease andendonuclease activities, and in 1976 the two activities wereattributed to the same enzyme, ExoIII [8,64]. ExoIII requires Mg2+

and is therefore EDTA-sensitive [65]. ExoIII is also heat sensitive, asit is inactivated rapidly upon incubation of the purified protein atmoderately high temperatures [66,67]. ExoIII possesses three othercatalytic activities in addition to its AP endonuclease activity. It is a30 to 50 exonuclease specific for dsDNA, allowing it to degrade bluntends, 50 overhangs or nicks to produce segments of ssDNA [68].

This activity is not observed on dsDNA with 30 overhangs that areover four bases long or on ssDNA [68]. ExoIII can also remove 30

phosphates to generate 30 OH termini, and has RNase H activitythat degrades RNA in DNA-RNA hybrids [68].

The hunt for a second AP endonuclease was initiated by thefinding that xth� mutants lacking ExoIII still possess �10% of thecell’s total AP endonuclease activity [66,67]. EndoIV was isolatedfrom crude extracts derived from these mutants [66,67]. Thisenzyme can incise the phosphodiester bond 50 of AP sites andcertain oxidized nucleotides (NIR described below), but not othertypes of damage (e.g. bulky lesions), to create single-strand breakswith 30 OH termini [66]. Like ExoIII, EndoIV possesses 30

phosphodiesterase activity allowing it to remove 30 blockinggroups at single-strand breaks such as those resulting from theactivity of AP lyases [69]. Purified EndoIV also possess 30 to 50

exonuclease activity with a preference for 30 recessed ends ofdsDNA [70]. In contrast to the AP endonuclease and 30 phosphodi-esterase activities, this exonuclease activity is inhibited in thepresence of the metal chelator EDTA and reducing conditions [70].So far, a biological role for the exonuclease activity of EndoIV hasnot yet been defined. It is noteworthy that EndoIV expression isinduced as much as 20-fold by superoxide anion generators such asparaquat [9]. As such, the level of EndoIV becomes comparable tothat of ExoIII, but its 30 diesterase activity always remains lowerthan that of ExoIII [9,71]. Similarly, expression of the human ExoIIIhomolog APE1 is increased by oxidative stress [72].

To date, E. coli EndoIV is the only member of the EndoIV familythat has been crystallized (Fig. 4A) [14,73]. The crystal structureshows that two of the Zn2+ ions coordinate a water molecule,which acts as the nucleophile to hydrolyze the DNA strand. Thethird Zn2+ ion stabilizes the resulting 30 OH. Mutation of the metal-coordinating residues impairs catalytic activity [73]. Otherunrelated nucleases utilize a similar mechanism to cleave DNA,but EndoIV is the only known DNA repair protein to employ thissystem [74,75]. Like many other DNA repair proteins, EndoIVrecognizes its substrate by flipping the AP site and its complemen-tary nucleotide out of the double helix (Fig. 4B) [14]. Instead ofkinking the DNA as shown for glycosylases, EndoIV bends the DNAat a 908 angle [14,76]. The increased flexibility of AP site-

[(Fig._2)TD$FIG]

Fig. 2. Chemical structures of EndoIV substrates, including (A) an AP site cleaved by the AP endonuclease activity, (B) a 30 phosphate blocking lesion removed by the 30

phosphodiesterase activity, and (C) cleavage upstream of a damaged base (a-20-deoxyadenosine is shown as an example) by the NIR activity.


containing DNA allows specificity for the target and prevents theenzyme from cleaving undamaged DNA [13]. Upon base flipping,EndoIV undergoes a conformational change that allows the highlyconserved residues Tyr72, Leu73 and Arg37 to form base stackinginteractions on either side of the AP site that stabilize the flipped-out conformation (Fig. 3, red arrows and Fig. 4C). Mutation of Tyr72to Ala decreases catalytic activity, but surprisingly does notprevent AP site flipping [73]. The space vacated by the flipped-outlesion (normally filled by Tyr72) is instead occupied by four watermolecules in the Tyr72Ala variant [73]. This suggests that Tyr72 isimportant for excluding solvent from the active site of the enzyme.

The active site can accommodate any base-free nucleotide,explaining the diverse phosphodiesterase activity of the EndoIVfamily on a variety of substrates.

In the ExoIII family, crystal structures have been obtained forboth E. coli ExoIII and human APE1 [68,77]. Comparison withEndoIV reveals that the two families utilize completely different,independently evolved structures to accomplish the same task.Whereas EndoIV uses a TIM (triosephosphate isomerase) barrelfold consisting of eight a-helices and eight parallel b-strands tointeract with DNA, ExoIII and APE1 use a 4-layered a,b sandwichfold [68]. Both enzymes rely on the increased flexibility of the AP

[(Fig._3)TD$FIG]

Fig. 3. Alignment of EndoIV family members across multiple species. Red arrows indicate conserved residues that form base stacking interactions to stabilize the flipped-out

AP site. Blue arrows indicate conserved amino acids that coordinate the Zn2+ ion.


site for recognition, however, and both ‘‘probe’’ the genome for APsites by inserting residues into the minor groove [13]. WhileEndoIV undergoes a striking conformational change upon AP sitebinding to accommodate the flipped-out nucleotide, APE1 doesnot; it is instead preformed to accept the AP site [77]. Bothenzymes cleave the DNA backbone by attacking the phosphodie-ster bond with a hydroxyl nucleophile, but different metals (Zn2+

for EndoIV and Mg2+ for ExoIII) are used for charge neutralization.Thus, the two families of enzymes represent a striking example ofconvergent evolution, in which the two enzymes evolved to share

many of the key aspects of the reaction despite starting from twocompletely different structures.

The relative contribution of ExoIII and EndoIV to repair in vivo

was investigated using mutants defective in either or both of thetwo enzymes. These studies revealed extensive overlap betweenthe two families [58]. Consistent with their respective activitiesin extracts, xth�mutants lacking ExoIII are very sensitive to MMSwhereas nfo�mutants devoid of EndoIV are only slightly sensitiveto the same drug [58,64]. Similarly, xth� cells are sensitive toH2O2, but nfo� strains show wild-type levels of resistance

[(Fig._4)TD$FIG]

Fig. 4. (A) Crystal structure of E. coli EndoIV in the absence of DNA. (B) EndoIV bound

to its product, with the 50 dRP that results from AP site cleavage highlighted in

yellow. (C) The AP site binding pocket of E. coli EndoIV.


[58,78,79]. xth� nfo� double mutants show increased sensitivityto both of these agents compared to single mutants, suggestingthat ExoIII is the primary enzyme that processes AP sites andstrand breaks with blocked 30 termini, with EndoIV playing abackup role [58]. This general trend does not apply to all types ofdamage, however, as nfo� mutants are highly sensitivity tobleomycin and tert-butyl hydroperoxide (t-BH) whereas xth�

mutant are only slightly affected by t-BH and not at all bybleomycin [58]. Furthermore, the double mutant shows no

increase in sensitivity to either of these agents when compared tothe single mutants [58]. These data suggest that these agentsinduce types of damage that can only be processed by EndoIV. Thespecific lesions responsible for these phenotypes are not known.Bleomycin produces oxidized AP sites, single-strand breaks with30 blocked ends, and double-strand breaks, while t-BH yieldsoxidized bases and likely other uncharacterized damage as well[80–82]. Oxidized bases are an intriguing candidate, as EndoIVhas endonuclease activity on these lesions (discussed below), butExoIII does not [1].

Besides its abasic endonuclease and 30 phosphodiesteraseactivities, EndoIV also possesses endonuclease activity whichcan cleave the DNA backbone immediately 50 of oxidative lesions ina process termed nucleotide incision repair (NIR) (Fig. 2C) [1,83].Notably, members of the ExoIII family lack this activity, with theexception of human APE1 [84]. This activity is able to cleaveupstream of several oxidized bases, including alpha-deoxyadeno-sine, 5-hydroxyuracil, 5,6-dihydrouracil and 5,6-dihydro-thymine,creating a 30 OH terminus which can then serve as a primer for DNAsynthesis and LP-BER [1,83]. The recognition of a wide variety ofoxidative lesions by EndoIV in addition to AP sites and 30 blockinggroups suggests that its active site is highly flexible. Thus, EndoIVcan participate in repair of oxidized bases either by performing NIRat the initial lesion or by cleaving the AP site generated byglycosylase-mediated removal of the base. In cell extracts, NIR andglycosylase cleavage appears to be utilized at approximately equalefficiencies [85]. However, the NIR activity may be favoured uponexposure to oxidants, as EndoIV expression is induced by agentsthat generate superoxide anions [9].

5. S. cerevisiae Apn1

The budding yeast AP endonuclease Apn1 was initiallyidentified as a 30 phosphodiesterase in crude extracts [3,15].Further characterization of the purified protein indicated that it isactive on a variety of 30 blocking lesions including phosphates,phosphoglycolates, aldehydes and deoxyribosephosphates, aswell as on AP sites [2,3,16]. Neither the 30 phosphodiesterase northe AP endonuclease activity showed any metal requirements.Apn1 also possesses NIR endonuclease activity and can cleave 30

phosphotyrosyl links that result from topoisomerase I trapping[1,86]. The biochemical properties of the yeast enzyme werereminiscent of E. coli EndoIV, which led to the notion that theenzymes are related [3,15,16]. When the APN1 gene wasidentified, sequence analysis indeed revealed that Apn1 shares41% identity with E. coli EndoIV [4]. Consistent with this,expression of ScApn1 in E. coli can substitute for the absence ofEndoIV [87]. To confirm that APN1 does indeed encode thepreviously identified AP endonuclease activity, further workshowed that apn1D extracts express <1% of the total 30

phosphodiesterase/AP endonuclease activity present in wild-type extracts, and overexpression of APN1 led to an �80-foldincrease in this activity [2,88]. Apn1 expression is not induced byexposure to oxidants, suggesting that this enzyme is not under aregulatory mechanism as observed for E. coli EndoIV.

apn1D cells devoid of Apn1 grow normally but show increasedsensitivity to MMS and the oxidants H2O2 and tert-butylhydroper-oxide (t-BH) [2]. DNA isolated from apn1 mutants treated withMMS or H2O2 shows an accumulation of unrepaired AP sites andDNA strand breaks, respectively, as compared to the wild-type [2].Thus, Apn1 is involved in repair of AP sites and strand breaks withblocked 30 termini in vivo. Surprisingly, apn1D mutants are notsensitive to bleomycin, which also induces strand breaks withblocked 30 termini [80,89,90]. This is likely because yeast containsother 30 phosphodiesterases that can efficiently remove the 30

phosphoglycolate ends induced by bleomycin (see below).


apn1D strains also accumulate spontaneous mutations at anincreased rate, and these mutations correspond mainly to singlebase-pair substitutions [31]. Since Apn1 has multiple enzymaticactivities and yeast does not seem to follow a simple ‘‘A-rule’’ likeE. coli, it was initially unclear whether these mutations arose fromunrepaired AP sites or other types of DNA damage processed byApn1, such as oxidative lesions or strand breaks with blocked 30

termini. Several independent investigations all indicated that thehigh spontaneous mutation rate observed in the absence of Apn1 isthe result of unrepaired AP sites. Simultaneous deletion of MAG1,the glycosylase responsible for converting 3-methyladenineresidues to AP sites, partially rescued the apn1D mutatorphenotype [31]. Expression of human APE1, which is a strongAP endonuclease but has weak 30 phosphodiesterase activity,reduced the mutation rate of apn1D yeast to wild-type levels [91].Finally, expression of E. coli EndoIII, an AP lyase that lacks 30

phosphodiesterase activity, also rescued the apn1D spontaneousmutation rate to wild-type levels [92]. This latter finding raised thequestion of how apn1D cells process the 30 a,b-unsaturatedaldehyde left by EndoIII, which was later explained by thediscovery of Apn2 (see below).

The identification of the APN1 gene allowed for analysis ofgenetic interactions between APN1 and other BER mutants. One ofthe first such interactions to be investigated was that with RAD27,which encodes the S. cerevisiae homolog of flap endonuclease 1(FEN-1). After Apn1 incises an AP site, Rad27 cleaves the 50 flapgenerated by displacing DNA synthesis during LP-BER. Surprising-ly, APN1 deletion was shown to rescue the MMS hypersensitivity ofa rad27D strain [93]. This indicates that the cell is better able totolerate intact AP sites than the strand breaks generated by Apn1incision.

The observation that extracts from apn1D cells still containdetectable AP endonuclease and 30 phosphodiesterase activitiessuggested the presence of a second AP endonuclease. This activitywas initially purified and characterized [10], and later the gene wasidentified as APN2, homologous to E. coli ExoIII [94,95]. UnlikeApn1, the 30 phosphodiesterase and exonuclease activities of Apn2are much stronger than its AP endonuclease activity, suggestingthat the two enzymes may have different substrate specifities in

vivo [96–98]. While deletion of APN2 alone produces no knownphenotype, apn1D apn2D double mutants are exquisitely sensitiveto MMS and H2O2, and show a higher mutation rate than that ofapn1D cells [94,95]. Thus, Apn1 appears to be the predominant APendonuclease/30 phosphodiesterase in yeast, with Apn2 playing abackup role that is only uncovered in the absence of Apn1. As in theapn1D background, MAG1 deletion partially rescues the mutatorphenotype of apn1D apn2D cells, highlighting the importance of APsites in mutagenesis [99].

Loss of the Rad1/Rad10 endonuclease, which is required fornucleotide excision repair, is lethal in the apn1D apn2Dbackground, indicating that Rad1/Rad10 provides backup activitythat enables the cell to survive spontaneous DNA damage in theabsence of the AP endonucleases [100,101]. The observations that(1) simultaneous deletion of all three AP lyases (NTG1, NTG2 andOGG1) allows apn1D apn2D rad1D cells to grow for severalgenerations before death and (2) UNG1 deletion rescues apn1Dapn2D rad1D lethality imply that endogenous AP sites, specificallythose generated by uracil excision, are the primary cause of deathin these cells [20,100].

The 30 DNA phosphatase Tpp1 also shows overlapping substratespecificity with Apn1. TPP1 encodes the yeast homolog ofmammalian polynucleotide kinase phosphatase (PNKP), but lacksthe 50 kinase domain present in the mammalian enzyme [102].Unlike Apn1 and Apn2, Tpp1 is specific for 30 phosphates and lacksgeneral 30 phosphodiesterase activity and AP endonuclease activity[103]. While tpp1D single mutants show no discernable pheno-

type, TPP1 deletion in an apn1D apn2D background causes severesensitivity to H2O2 and bleomycin, which induce strand breakswith 30 phosphates, but not MMS [103]. In addition, apn1D tpp1Drad1D cells grow slowly due to spontaneous oxidative damage[104]. The slow growth of this strain can be rescued by deletion ofgenes involved in the anaphase-metaphase checkpoint, but at theexpense of increased chromosome instability [105]. Collectively,these data show that yeast uses multiple overlapping methods,including Apn1, to repair strand breaks with 30 phosphate termini.

Apn1 can also remove 8oxoG from the 30 end of a DNA strandusing its 30 exonuclease activity, a situation likely most relevant tomisincorporation of 8oxoG from the dNTP pool during DNAsynthesis [106]. Deletion of APN1 in an ogg1D background causes a46-fold increase in the spontaneous mutation rate, indicating thatthis activity helps to promote faithful DNA synthesis [106]. The 30

exonuclease activity of Apn1 may be particularly important in S.

cerevisiae because yeast lacks a homolog of E. coli MutY, whichremoves adenine misincorporated opposite 8oxoG during DNAsynthesis [107,108].

In Apn1, mutation of the conserved glutamic acid residue 158 toglycine drastically reduces the catalytic activity of the enzyme buthas no effect on DNA binding [109]. In contrast, the correspondingE145G mutation in EndoIV prevents the protein from binding toDNA [110]. E145 of EndoIV makes contact with one of the Zn2+ ionsin the active site and is located close to one of the R-loops(containing residues 149–153) involved in DNA binding [14]. Thus,the discrepancy could be explained by a more severe conforma-tional change induced by the glycine mutation in EndoIV than inApn1, perhaps disrupting the position of the nearby R-loop.

5.1. The role of Apn1 in mitochondrial BER

The mitochondrial genome is perhaps more susceptible to DNAdamage than the nuclear genome because of its close proximity toreactive oxygen species generated during aerobic respiration. Inrecent years, Apn1 and a number of other nuclear BER proteinswere found to be localized to the mitochondria [111–117].Mitochondrial localization of Apn1 is mediated by Pir1, a proteininitially described as a cell wall constituent of unknown function[112]. Pir1 binds to the C-terminus of Apn1, a region which alsocontains a nuclear localization signal, suggesting that Pir1 mayfunction by competing directly with the nuclear transportmachinery [88,112]. Deletion of PIR1 eliminates mitochondriallocalization of Apn1 and causes a corresponding increase innuclear Apn1, and strains lacking either Pir1 or Apn1 showincreased mitochondrial mutagenesis upon MMS treatment[112,118]. The N-terminus of Apn1 contains a putative mitochon-drial localization signal, but the requirement for Pir1 for propermitochondrial targeting indicates that this signal is not sufficientfor Apn1 mitochondrial targeting. Whether this region is necessaryfor Apn1 mitochondrial targeting or sufficient to target acytoplasmic protein to the mitochondria has not yet been tested.Interestingly, Apn1 overproduction causes increased mitochon-drial genome instability, likely by converting AP sites and oxidizedbases into more toxic strand breaks [119]. Thus, the import of Apn1into the mitochondria must be tightly regulated to ensure that theenzyme is present at optimal levels.

6. S. pombe Apn1

Fission yeast has an EndoIV homolog, Apn1 (referred herein asSpApn1), but unlike in S. cerevisiae, it does not seem to play a majorrole in AP site repair [6]. SpApn1 is expressed at very low levels andS. pombe apn1D mutants are not sensitive to MMS [120]. Instead,fission yeast relies on other repair enzymes such as Uve1 and theAP lyase Nth1 to cleave AP sites [60,120]. In the latter case, the


resulting 30 a,b-unsaturated aldehyde is believed to be removedby SpApn2 [59]. Overexpression of SpApn1 in Spapn2D cellspartially restores MMS sensitivity, indicating that SpApn1 can actas a 30 phosphodiesterase in this organism, although less efficientlythan SpApn2 [121]. Despite this phenotype, extracts from cellsoverexpressing SpApn1 show no ability to incise AP sites or remove30-blocking groups at DNA single-strand breaks [6]. Also, unlike E.

coli EndoIV or C. elegans APN-1 (see below), SpApn1 expression in S.

cerevisiae failed to produce a functional protein or restore MMSresistance to the apn1D apn2D double mutant (Ramotar, unpub-lished). One possible explanation for the reduced activity ofSpApn1 is that the protein contains an insertion of 24 amino acidsin the region surrounding one of the conserved His residues thatcoordinates the Zn2+ ion (Fig. 3). This stretch of residues is notpresent in any other species sequenced to date and might decreasethe catalytic activity of the protein, although the effect of deletionof this region has not yet been tested. Whether SpApn1 might beactivated by post-translational modifications under specific stressconditions also remains unexplored. Thus, the role of the EndoIVfamily member in fission yeast remains enigmatic.

7. C. elegans APN-1

The key role that the E. coli and S. cerevisiae AP endonucleasesplay in the base excision repair pathway led to the assumption thatproteins of these families might be conserved in higher organisms.The high homology seen between E. coli endonuclease IV and S.

cerevisiae Apn1 was used to isolate the cDNA of the C. elegans apn-1

gene using a l phage cDNA library prepared from C. elegans [122].The C. elegans protein shares 40.4% identity with S. cerevisiae Apn1and 44.9% identity with E. coli EndoIV [122]. Furthermore, crudeextracts from C. elegans embryos were found to possess APendonuclease activity [123]. However, it was uncertain whetherthis activity could be accounted for by the isolated apn-1 gene or toanother AP endonuclease present in this organism. Later experi-ments showed that two C. elegans genes, apn-1 and exo-3, encodingmembers of the EndoIV and ExoIII families respectively, couldcomplement the MMS and H2O2 sensitivity of apn1D apn2D yeastand restore AP endonuclease activity in extracts [124]. While exo-3

restored full resistance to bleomycin, apn-1 conferred only partialresistance to this drug, suggesting that the substrate specificity ofthe two enzymes differs [124]. Studies in progress have revealedthat APN-1 has NIR and 30–50 exonuclease activities, like itsbacterial and yeast counterparts (Yang and Ramotar, unpublished).The latter activity may play a more prominent role in C. elegans

than in other model organisms because EXO-3 lacks 30 exonucleaseactivity and C. elegans lacks an OGG1 homolog [124,125]. Thus, thisorganism may rely exclusively on the 30 exonuclease activity ofAPN-1 to remove misincorporated 8oxoG from the dNTP pool.

RNA interference experiments further revealed the biologicalimportance of C. elegans apn-1 in vivo. A 5-fold increase in thefrequency of spontaneous frameshift mutations was observed inapn-1 knockdown animals [5]. These worms were also sensitive toMMS and H2O2 [5]. A delay in the division of the P1 blastomere, theposterior cell of the two-cell embryo known to possess an activecell cycle checkpoint, was also detected following apn-1 knock-down, suggesting accumulation of unrepaired lesions [5]. Longev-ity of MMS and t-BH-treated worms was also reduced followingapn-1 RNAi [5]. apn-1 knockdown also rescued the lethality of dut-

1 knockdown worms, in which increased dUTP pools lead to uracilmisincorporation into DNA and formation of AP sites by uracil DNAglycosylase action [5]. These data indicate that APN-1 contributesto DNA repair in vivo, and, like in yeast, strand breaks generatedupon cleavage of AP sites are more toxic than AP sites in C. elegans.

It is noteworthy that C. elegans APN-1 has an extended N-terminus consisting of 118 amino acid residues (Fig. 3). Three other

species (Neurospora crassa, Aspergillus nidulans, and Strongylocen-

trotus purpuratus) also possess N-terminal extensions, but theseregions share little homology (Fig. 3). The biological function ofthese extensions is unknown, as the C. elegans N-terminus is notrequired for complementation of S. cerevisiae apn1D apn2Dmutants [124]. To test the possibility that this region could carrycryptic mitochondrial or nuclear targeting signals, we micro-injected a plasmid expressing the C-terminal extension fused toGFP into C. elegans, but no positive transgenic animals wererecovered (Ramotar, unpublished). It is therefore possible thatoverexpression of this fragment is toxic.

8. Conclusions and future directions

It is clear from the data discussed here that the APendonucleases are endowed with a variety of nucleolytic activitiesand are involved in BER and related pathways at multiple steps.Recent work has begun to illuminate how each activity of the APendonucleases is utilized at different types of lesions and repairintermediates, and how these enzymes function in concert withthe other players in the DNA damage response. These versatileenzymes are of tantamount importance in protecting the cellagainst endogenous DNA damage, as highlighted by the lethality ofAPE1 knockdown in human cells [126].

A major outstanding question in the field is why EndoIVhomologs were lost in higher organisms in favour of ExoIII familymembers. The observation that APE1, the ExoIII homolog inhumans, has NIR endonuclease activity, but S. cerevisiae Apn2 doesnot suggests that ExoIII family members gained NIR activity atsome point in evolution. Perhaps selective pressure for retention ofEndoIV homologs was lost when the earliest ExoIII homolog gainedthis activity. The importance of the NIR activity of EndoIV has beenshown using separation of function mutants that retain APendonuclease activity but lack NIR activity [127]. These mutantsconfer resistance to MMS, but not t-BH, indicating that oxidativelesions are indeed processed by NIR in vivo [127]. This hypothesiscould be examined by purifying and testing representative APendonucleases for NIR activity in some of the intermediate speciesfor which this activity has not yet been explored. Comparing therelative contributions of EndoIV and ExoIII family members in thehighest organisms which still retain both, such as C. elegans, X.

tropicalis and D. rerio, could also provide insight into whether ExoIIImembers do indeed account for the majority of repair activity inhigher organisms as observed in S. pombe.

It is also possible that EndoIV members do indeed exist inhigher organisms but are substantially diverged and thereforecannot be detected by standard bioinformatic methods. This isunlikely, however, because loss of APE1 in human cells causes celldeath. Expression of S. cerevisiae Apn1 in APE1-deficient cellsrestores survival, indicating that the observed lethality is due tothe accumulation of DNA damage and suggesting that APE1 is thesole AP endonuclease/30 phosphodiesterase [126]. An auxiliarypathway that can process AP sites in human cells has also beendescribed in which DNA glycosylases with AP lyase activity such asNEIL1 and NEIL2 cleave the AP site, leaving a 30 phosphate that canbe processed by the 30-phosphodiesterase activity of PNKP [128].The fact that APE1 deficiency causes lethality indicates that thisauxiliary pathway alone is unable to handle endogenous damageincurred by the cell.

Two additional approaches also failed to reveal a human EndoIVhomolog. Purification for a Mg2+-independent AP endonucleaseactivity from human cells led to the identification of GAPDH as abinding partner for APE1 [129]. This complex has a low level ofactivity in the absence of Mg2+ as a result of APE1 and not amember of the EndoIV family that was sought [129]. In anotherapproach, we raised an antibody against a synthetic peptide


corresponding to the most conserved region of the EndoIV familyof enzymes, but it failed to detect a human EndoIV protein in cellextracts, although an immunoreactive polypeptide has beenobserved to copurify with AP endonuclease activity from calfthymus (Ramotar, unpublished).

Finally, an intriguing question that remains largely unexploredis to determine how AP endonucleases access DNA lesions in thecontext of chromatin and a milieu of other DNA binding proteins,such as those involved in transcription and the DNA replicationmachinery. Specific histone modifications may serve to recruit orstimulate AP endonucleases, as has been demonstrated for theDNA glycosylase MBD4, which is more efficient at removing T:Gmismatches upon histone acetylation [130]. Such marks couldmodulate the relative usage of EndoIV and ExoIII family membersat specific lesion types, for example, bleomycin-induced strandbreaks with blocked 30-termini.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

J.M.D. was supported by a post-doctoral fellowship from the LeFonds Quebecois de la Recherche sur la Nature et les Technologies.This work was supported by funding from the Natural Sciences andEngineering Council of Canada (grant # 202432-01) to D.R.

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