drosophila blm in double-strand break repair by synthesis...
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Supporting Online Material
Drosophila BLM in Double-Strand Break Repair
by Synthesis-Dependent Strand Annealing
Melissa D. Adams1*, Mitch McVey1,2*, Jeff J. Sekelsky1,3†
* These authors contributed equally to this work.
1Department of Biology, 2SPIRE Program, and 3Program in Molecular Biology and
Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
† To whom correspondence should be addressed. E-mail: [email protected]
Material and Methods
Drosophila Stocks and Genetics
Flies were maintained on standard medium at 25οC. The P{wa} stock (S1) contains a 14-kb P
element that carries the apricot allele of the white gene (wa). In the wa allele, a 5 kb copia
retrotransposon is inserted into the second intron of white. The copia element has directly
repeated 276 bp long terminal repeats (LTRs) at each end. Inverse PCR was used to map the
P{wa} insertion site to an intron of scalloped. The mus309 mutants used in these studies were
compound heterozygotes containing the mus309D2 and mus309D3 alleles (S2). The stable
transposase source P{ry+, ∆2-3}99B is described in (S3).
Analysis of DNA Repair Synthesis
Genomic DNA was prepared from single flies according to (S4). PCR reactions contained 10
mM Tris-HCl pH 9.0, 50 mM KCl, 2.5 mM MgCl2, 0.1% Triton X-100, 1.25 µM each primer,
250 µM dNTPs, 2 µl of the genomic DNA prep and TAQ polymerase in a 20 µl volume. PCR
products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining.
Gel-purified PCR products were sequenced directly.
Discussion
Flanking deletions formed in mus309 mutants
Beall and Rio (S6), using a plasmid injection assay, reported frequent flanking deletions
associated with DSB repair in mus309 mutants. In our assay, the DSB left after excision of
P{wa} is within an intron of an essential gene, sd, and deletions that extend to an exon (3.6 kb to
the left or 1.7 kb to the right) result in lethal sd alleles. Among 71 aberrant repair events
generated in wild-type males, only 1 had a lethal mutation. This corresponds to 0.06% of total
progeny (1.5% of yellow-eyed progeny, which constitute 4.2% of total progeny). The generation
of large deletions is therefore a somewhat rare event in wild-type flies. In contrast, 40 of 147
aberrant repair events generated in mus309 mutant males had lethal sd mutations. This
corresponds to 3.8% of total progeny, and represents a 63-fold increase over the rate observed in
wild-type males. P element excision is commonly used by Drosophila geneticists to make
deletions flanking a P element insertion site (reviewed in S6). It is clear from our results that
large deletions are produced much more frequently mus309 mutant males. The use of mus309
mutants in P element excision schemes should greatly increase the probability of recovering
flanking deletions.
Aborted SDSA events
Aborted SDSA events result in internally deleted P elements. Failure of SDSA in mus309
mutants can be attributed to defects in repair DNA synthesis and/or strand invasion. The basis
for SDSA failure in wild-type flies is less clear. In our assay, aberrant repair events generated by
wild-type males usually involved long stretches of repair synthesis. Although this suggests that
defects in repair synthesis are not the cause of SDSA failure in wild-type flies, it is possible that
some sequences or chromatin domains are difficult for the repair synthesis machinery to traverse.
However, we did not observe any clustering of junction points, either in wild-type or in mus309
mutants (table S2).
Another possibility is that SDSA proceeds until a critical phase of the cell cycle is reached,
such as entry into mitosis, at which point SDSA is aborted in favor of end-joining pathways.
SDSA across a gap may involve repeated rounds of strand invasion and repair DNA synthesis
(S7). If complementary sequences have not been synthesized by the time a critical phase of the
cell cycle is reached, a DNA damage checkpoint may be invoked, with one consequence being to
prevent new strand invasions in favor of end-joining pathways.
Fig. S1. Isolation of DSB repair events that occur subsequent to P element excision. (A) The
cross performed to recover DSB repair events following excision of P{wa} is shown (only
markers relevant to the experiment are indicated). Mitotic germline repair events were recovered
by crossing single males containing an X-linked copy of the P{wa} element and an autosomal
copy of the stable P transposase source P{ry+ ∆2-3} to homozygous w P{wa} virgin females.
Square brackets indicate that the males were either completely wild-type for mus309 (+/+), or
were heterozygous for two independently isolated mus309 alleles (mus309D2/mus309D3). Sb+
female progeny that inherited the paternal X chromosome containing a potential repair event,
P{?}, were scored for eye color, which is indicative of the type of repair event recovered. (B)
Somatic excision of P{wa} leads to eye color mosaicism in the males described in A. Patches of
red pigment, which arise by repair through an SDSA pathway, are dramatically reduced in
mus309 mutant flies. Somatic mosaicism was dependent on the presence of P transposase.
X w P{wa}
w P{?}
w P{wa};
+
[mus309D3]
A
Bwild type mus309
w P{wa}
Y;
[mus309D2] Sb P{ry+ ∆2-3}[mus309D3]
somatic and germline excisions
mosaic eyes
score eye color
Fig. S2. Isolation of DSB repair events for molecular analysis. To characterize aberrant repair
events, yellow-eyed female progeny from the cross described in fig. S1 were crossed to males
containing an X chromosome balancer. Each of these yellow-eyed females was isolated from a
separate vial (see table S1) and thus represented an independent repair event. The P{?}
chromosome was recovered in white-eyed male offspring to facilitate molecular analysis. The
absence of white-eyed sons was taken to indicate that a particular repair event was associated
with a lethal mutation. In this case, molecular analysis of P{?} was performed with DNA from
balanced females (P{?}/FM7w). (That the white allele on the balancer chromosome is w1, not
wa.) We verified that lethality was associated with loss of sd gene function by crossing
P{?}/FM7w females to sd1 (S8) males and scoring for a scalloped-wing phenotype.
X FM7w Y
w P{?}
w P{wa};
+
[mus309D3]
w P{?}
Y
w P{?}
FM7w
yellow eyes
recover viable eventsin males
recover lethal eventsin females
Fig. S3. Analysis of repair DNA synthesis. (A) Genomic DNA from white-eyed male offspring
described in Fig. S2 was analyzed by PCR. The left and right ends of the break remaining after
P{wa} excision are drawn. Primers specific for the P element terminal inverted repeat (black
rectangles) and the flanking sd intron (blue) were used to determine whether repair synthesis had
initiated from the broken chromosome ends. Only ten 3’ terminal nucleotides of the P-specific
primer are complementary to the overhang remaining after P element excision (dotted line).
Thus, extension of the primer under our PCR conditions requires repair synthesis from the end of
the break. The ability to amplify DNA from each end of the double-strand break site therefore
correlates with the presence of repair synthesis. Both the left and right ends of each P excision
site were analyzed in this manner for wild type and mus309 flies. (B) The right end of the P{wa}
element is shown. Primer pairs spanning the P{wa} element (A-D) were used to analyze the
extent of repair synthesis from the end of the DSB in flies that had undergone aberrant DSBR.
The ability to specifically amplify P{wa} sequences correlates with repair synthesis and
delineates a minimum amount of DNA synthesized. For example, amplification of P{wa}
sequence with primer pair B, but not primer pair C, indicates repair synthesis of at least 0.9 kb.
B
A
Left End
Right End...
...DSB
Right End
A
B
C
D
0.9 kb2.4 kb4.6 kb >5 bp
Table S1. Distribution of progeny classes from independent crosses. Single males with P{wa}
and P transposase were crossed to three w P{wa} virgin females in vials (fig. S1). The number
of independent crosses analyzed for wild type and mus309 mutants is given. Comparison of the
number of productive vials, meaning those which produced progeny, and the number of Sb+
female progeny demonstrates that there was no decrease in male fertility in the
mus309D2/mus309D3 background. The number and percentage of productive vials that gave rise
to at least one red-eyed daughter, or at least one yellow-eyed daughter are given. Among total
Sb+ female progeny of mus309 males, red-eyed flies were reduced by a factor of 38, relative to
the progeny of wild-type males (see text and Fig. 1). As shown here, there was a similar
decrease (by a factor of 30) in the number of individual males that produced at least one red-eyed
daughter.
Genotype Vials Productive vials
Sb+ female progeny
Sb+ female progeny per vial
Vials with red-eyed progeny
Vials with yellow-eyed progeny
wild-type 274 246 (90%) 6263 25 148 (60%) 143 (58%)
mus309 164 154 (94%) 5318 35 3 (2%) 148 (96%)
Table S2. Junction sequences of repair products generated in the presence or absence of
DmBlm. See Table 1 in the text for details.
Isolate Boundaries Sequence
Class 1: junctions with microhomologies
WT1 5475 / 2532 GAGGCTGCTACTGAG:TTCT:TCTAGCCACTCAGTG
WT2 2098 / 2685 CATTTGAGCGAACCG:AAT:TTATTTTTCAAAACG
WT3 1909 / 2572 CTTTCAGTTCAAATT:G:CTTCACTGCTCATCT
WT4 -1 / 2 ATATCATACCCCTGC:TGACCCAGAC:TCTCACTTGACGCC
WT5 2876 / 735 AGCGCCATCGAGGTC:GA:TGGCGTAAACCGCTT
WT6 12 / 136 AGACCATGATGAAAT:A:TCAACAATCATATC
WT7 17 / 280 CATGATGAAATAACA:TA:TAGGGGGATGGGAAT
M1 15 / 57 CCATGATGAAATAAC:A:GCATACGTTAAGTGG
M2 205 / 5 CTGGAGTAAAATTAA:TTCA:TCATGACCCAGACTC
M3 -1289 / 112 ATACTAAACATATTG:TCA:CTCAGACTCAATACG
M4 624 / 343 ATTTCATACGTTACT:GATAT:GCAAATCGTACTCAC
M5 100 / 43 TTGAGAGGAAAGGTT:GTG:GATGTCTCTTGCCGA
M6 5 / 150 GCTGACCCAGACCAT:GA:CATGCTAAGGGTTAA
M7 28 / 260 AATAACATAAGGTGG:TCCCG:GGGATCCGTCGACCT
M8 1425 / 3558 AAAAAAAGACCGCAA:CAA:GGTTCCTCCACCATG
M9 537 / -451 AATAAGAGCTTGAGG:GA:TATGTTTGACTGAAA
M10 1201 / -638 TATTATTATTATTTT:TA:AAGCGTTGAACATTT
M11 59 / 225 CTTACCGAAGTATAC:ACT:CGCAAATTATTAAAA
M12 412 / -1 CCAGAAAAGATAAAA:GA:CCCAGACTCTCACTT
Isolate Boundaries Sequence
M13 415 / -1514 GAAAAGATAAAAGAA:GG:GATATTTTCGATAGT
M14 171 / -1084 GAAATATTGCAAATT:TT:AATGCCGTTCAACCC
M15 502 / -2943 GTGACTGTGCGTTAG:GT:GATGCGCACTTAGTG
M16 85 / 1088 CACGTTTGCTTGTTG:AG:CTACCAGAATAATCT
M17 521 / -1 CCTGTTCATTGTTTA:ATGAA:CCCAGACTCTCACTT
M18 206 / -903 AGTAAAATTAATTCA:C:ATTGCAGTCTGGCTT
Class 2: junctions without microhomologies
WT8 5634 / 5631 ACCGCTACCGTCGAC:GAATTTCCCTTGAAT
WT9 3905 / 5176 ATGTAATGCTAGATA:ATAAGTTCGTCAAAA
WT10 1394 / 2594 GTTGCCACGTTGGAA:TTGCATTTCCTCCTT
M19 131 / 16 TTTGAAAACATTAAC:ATGTTATTTCATCAT
M20 189 / 116 GCAAAGCTGTGACTG:TCACTCAGACTCAAT
M21 400 / 1134 AGAGCCTGAACCAGA:TCTTGATCATGATAT
M22 -82 / 32 CCTTTACCTATGCTA:CCGACGGGACCACCT
M23 15 / 437 ATGATGAAATAACAA:CGAACCAACGAGAGC
M24 131 / 16 TTTGAAAACATTAAC:ATGTTATTTCATCAT
M25 -1 / -93 CCTGCTGACCCAGAC:AGGGTATGTGCCACA
Class 3: junctions with insertions
WT11 3191 / 5367 CAAATGTATTCTAAA:tgaacatga*:CGTTGTGGTCATTTT
WT12 2561 / 287 CTCCAGGATGACCTT:ctattctagg:GGGATTCTAGGGGGA
WT 13 12 / 258 GACCATGATGAAATA:tc:GATCCGTCGACCTGC
WT 14 14 / 13 CCATGATGAAATAAC:ttctaacttataattatatataac
ttataacttataac†:TTATTTCATCATGAC
Isolate Boundaries Sequence
M26 53 / 141 AAGCTTACCGAAGTA:atcaa:GGTTAATCAACAATC
M27 66 / -2069 TATACACTTAAATTC:tttt:TATTCTTTTTTTTTT
M28 217 / -893 TCACGTGCCGAAGTG:ccgaag:TTGAAAAACCATTGC
M29 17 / 436 TGATGAAATAACATA:ttt:CCGTTTACTGTGTGA
M30 1049 / 2832 GGCAAACTCCTTATT:c:GGCAAAATCCGAAGA
M31 -2 / 5 CCCTGCTGACCCAGA:ggg:TCATGACCCAGACTC
* A possible template for this insertion is located 67 bp to the left of the junction. † The de novo sequence addition in this junction contains several repeats of the underlined P
element sequence.
Supplemental References
S1. M. Kurkulos, J. M. Weinberg, D. Roy, S. M. Mount, Genetics 136, 1001 (1994).
S2. K. Kusano, D. M. Johnson-Schlitz, W. R. Engels, Science 291, 2600 (2001).
S3. H. Robertson et al., Genetics 118, 461 (1988).
S4. G. B. Gloor et al., Genetics 135, 81 (1993).
S5. E. L. Beall, D. C. Rio, Genes Dev. 10, 921 (1996).
S6. M. D. Adams, J. J. Sekelsky, Nat. Rev. Genet. 3, 189 (2002).
S7. F. Pâques, W.-Y. Leung, J. E. Haber, Mol. Cell. Biol. 18, 2045 (1998).
S8. D. L. Lindsley, G. G. Zimm, The Genome of Drosophila melanogaster (Academic Press,
Inc., San Diego, CA, 1992).