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: sekelsky@unc.edu

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).