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. 2006 Nov 7;103(45):16840-5.
doi: 10.1073/pnas.0607904103. Epub 2006 Oct 30.

Template disruptions and failure of double Holliday junction dissolution during double-strand break repair in Drosophila BLM mutants

Dena Johnson-Schlitz  1 , William R Engels
Affiliations

Template disruptions and failure of double Holliday junction dissolution during double-strand break repair in Drosophila BLM mutants

Dena Johnson-Schlitz et al. Proc Natl Acad Sci U S A. .

Abstract

Previous biochemical studies of the BLM gene product have shown its ability in conjunction with topoisomerase IIIalpha to resolve double Holliday structures through a process called "dissolution." This process could prevent crossing over during repair of double-strand breaks. We report an analysis of the Drosophila BLM gene, DmBlm, in the repair of double-strand breaks in the premeiotic germ line of Drosophila males. With a repair reporter construct, Rr3, and other genetic tools, we show that DmBlm mutants are defective for homologous repair but show a compensating increase in single-strand annealing. Increases of 40- to 50-fold in crossing over and flanking deletions also were seen. Perhaps most significantly, the template used for homologous repair in DmBlm mutants is itself subject to deletions and complex rearrangements. These template disruptions are indicative of failure to resolve double Holliday junctions. These findings, along with the demonstration that a weak allele of topoisomerase IIIalpha has some of the same defects as DmBlm, support the dissolution model. Finally, an analysis of DmBlm mutants in conjunction with mus81 or spnA (Rad51) reveals a second function of BLM distinct from the repair of induced double-strand breaks and possibly related to maintenance of replication forks.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Tests of DSB repair with Rr3. (A) DSB repair pathways are measured in the germ line of individual males. In this example, the test males are null for DmBlm. All test males are heterozygous for the reporter construct, Rr3 (24). This construct includes a nonfunctional copy of the DsRed gene that is interrupted by a recognition site (●) for the I-SceI endonuclease. It also contains a 147-bp duplication, symbolized by the letters sRe within DsRed. The other homolog carries a modified version of Rr3, called Rr3EJ1, in which the I-SceI cut site has been destroyed by the insertion of 14 bp and loss of 2 bp (X). It also has a 16-bp deletion (∗) relative to Rr3 located 156 bp to the right of the former cut site. Heterozygous visible markers aristaless (al) and speck (sp) flank Rr3. The offspring from each test male are classified and subclassified as shown to obtain the numbers N1–N7. For example, N1 is the total number of offspring, N2 is the number of recombinants, N3 is the number of females. Estimates of N6 and N7 required PCR tests of a sample of 10 DsRed− individuals, or as many as were available. (B) Averages and standard errors obtained from multiple independent test males as shown in A. Results show that mutations at either DmBlm or Top3α result in increases in recombination and SSA while decreasing HR-h. The numbers of test males for the four genotypes shown were (left to right) 115, 139, 91, and 46. The numbers of offspring scored were (left to right) 7,878, 20,282, 14,052, and 2,211. Results for WT (+/+) are from table 2 of Preston et al. (24). Allele abbreviations are as follows: D2, mus309D2; T7, Df (3)T7; top, Top3αEP2272. (C) DmBlm and Top3α mutations increase flanking deletion frequencies estimated from the same progeny as in A and B by scoring the number of progeny with complete or partial loss of mini-white (w*) expression among all progeny with phenotype al DsRed− sp. The numbers of test males and allele abbreviations are as in B.
Fig. 2.
Fig. 2.
Tests of mus81 phenotypes. (A) Synthetic lethality of mus81 and mus309. Survival frequency is measured from the numbers of Tubby and Tubby+ offspring from a cross in which both parents were homozygous (or hemizygous) for a null allele of mus81 on the X chromosome and heterozygous for null alleles of DmBlm on chromosome 3. The other third chromosome was TM6B, which has an embryonic recessive lethal and the dominant visible marker, Tubby. The latter can be identified at larval, pupal, and adult stages. Survival frequencies were corrected for Mendelian expectations. See Flybase (31) for genetic symbols. There were 131 total offspring scored at the third-instar larval stage, 105 pupae, and 72 adults. (B) There was no apparent effect of mus81 on any of the measures related to DSB repair. Data were obtained by a scheme analogous to that shown in Fig. 1A except that the endonuclease was on a third chromosomal balancer. “Short HR-h” refers to the length of the conversion tract and is defined operationally in Methods. The averages and standard errors were computed from a number of independent estimates ranging from 61 to 77 for SSA, NHEJ, HR-h, recombination, and deletions. For short HR-h, there were 43 and 49 independent measurements for WT and mus81, respectively.
Fig. 3.
Fig. 3.
DSB formation opposite a large insertion. (A) Double-stranded 54-mer used to replace P{w*} insertion at cytological position 50C. The I-SceI recognition sequence flanked by 17-bp P element termini was inserted into chromosome 2 at the identical position as a 5-kb P element insertion (see Methods). (B) Genotype of parental males used in C and Fig. 4. The I-SceI endonuclease, if present, is on the X chromosome. The visible markers, al, b, cn, and bw (31) are close to the tips and bases of the two arms. P{w*} indicates the 5-kb P{CaSpeR}Cp150C insertion, which carries a mini-white gene (31). “Cut Site” refers to the I-SceI recognition site and surrounding sequence shown in A. The marker sites located 2.3 kb to the left of the insertion point (L) and 1.4 kb to the right (R) were distinguishable between the homologs by use of PCR as described by Preston et al. (44). (C) Recombination frequencies in the indicated interval in the premeiotic germ line of males as shown in B. Genotypes at the DmBlm locus were either heterozygotes (mus309D2/+), abbreviated “D2/+,” or null mutants (mus309D2/Df(3)T7), abbreviated “D2/T7.” Categories of I-SceI endonuclease are as follows: absent (“−”), maternal effect only (“M”), combined maternal effect and zygotic on the X chromosome (“MZ”), or zygotic X chromosomal only (“Z”). ND, not done. Each frequency is the average of multiple independent measurements from individual males. The numbers of males used for al-b recombination were (top to bottom) 71, 118, 159, 156, 195, 100, 111, 96, 72, 96, and 39 with a total of 76,215 progeny scored. For the cn-bw interval, the numbers of males were 75, 84, 68, 81, 81, 77, 85, 58, 60, 68, 67, and 41 with a total of 113,331 total progeny.
Fig. 4.
Fig. 4.
HR-h and template disruptions. (A) HR-h and template disruptions were measured by the scheme in Fig. 3 A and B. HR-h was computed as the proportion of offspring expressing w* among those receiving the cn+ bw+ chromosome. The template disruption frequency was defined as the proportion of offspring lacking w* expression among those receiving the cn bw chromosome. As described in Methods, these estimates required test-crossing a sample of candidates from each male's offspring. DmBlm genotypes and endonuclease sources are as in Fig. 3C. The effect of DmBlm mutations to reduce HR-h and increase template disruptions were highly significant. The numbers of single-male crosses used to estimate HR-h were (top to bottom) 46, 116, 155, 148, 187, 96, and 107, and for template disruptions, they were 71, 117, 152, 155, 188, 100, and 105. (B) Maps of a sample of nine template disruptions were obtained by a combination of DNA sequencing, PCR amplifications with primers within P{CaSpeR} and in the 50C region, and complementation tests with known lethal and sterile mutations in the vicinity. Genetic symbols are defined in FlyBase (31). Sequence differences were used as in ref. to distinguish regions derived from the P{CaSpeR} chromosome, which carried PCR markers LC and RC, versus its homolog with the I-SceI cut site and markers LA and RA (see Fig. 3B). Dashed brackets indicate approximate endpoints. The structure in TD1 was identical to the I-SceI cut site sequence shown in Fig. 3A except for several altered bases (“x”) typical of NHEJ products.
Fig. 5.
Fig. 5.
Male recombination frequencies for mutation combinations. Comparison of the first two rows shows that loss of the rad51 function abolishes the recombination induced by lack of DmBlm. The other rows confirm the effect on recombination by DmBlm and Top3α. The heterozygous controls for DmBlm were pooled from data by using mus309D2/+ and Df(3)T7/+ that yielded indistinguishable results. The DmBlm mutants were mus309D2/Df(3)T7. The Top3α genotypes were as in Fig. 1, and the genotypes for DmRad51 were spnA1/+ and spnA1/spnA1.
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