Multiple functions of Drosophila BLM helicase in maintenance of genome stability

Abstract

Bloom Syndrome, a rare human disorder characterized by genomic instability and predisposition to cancer, is caused by mutation of BLM, which encodes a RecQ-family DNA helicase. The Drosophila melanogaster ortholog of BLM, DmBlm, is encoded by mus309. Mutations in mus309 cause hypersensitivity to DNA-damaging agents, female sterility, and defects in repairing double-strand breaks (DSBs). To better understand these phenotypes, we isolated novel mus309 alleles. Mutations that delete the N terminus of DmBlm, but not the helicase domain, have DSB repair defects as severe as those caused by null mutations. We found that female sterility is due to a requirement for DmBlm in early embryonic cell cycles; embryos lacking maternally derived DmBlm have anaphase bridges and other mitotic defects. These defects were less severe for the N-terminal deletion alleles, so we used one of these mutations to assay meiotic recombination. Crossovers were decreased to about half the normal rate, and the remaining crossovers were evenly distributed along the chromosome. We also found that spontaneous mitotic crossovers are increased by several orders of magnitude in mus309 mutants. These results demonstrate that DmBlm functions in multiple cellular contexts to promote genome stability.

Figure 1.—

mus309 alleles. Boxes indicate exons; untranslated regions are hatched (only the beginning of the 3′-UTR is shown) and the region encoding the RecQ core is shaded. Vertical lines mark the positions of the seven conserved motifs of superfamily II helicases. The insertion site of the P{EPgy2}mus309^EY03745 element used to generate deletions is indicated by a solid triangle. The positions of the nonsense mutation in mus309^D2 and the missense mutation in mus309^D3 are given above the schematic, and the regions deleted in mus309^N alleles are indicated below with dashed lines.

Figure 2.—

Phenotypes of embryos from mus309 mutant females. Representative DAPI-stained syncytial-stage embryos from wild-type (w¹¹¹⁸) or mus309 mutant females are shown. Defects observed frequently include anaphase bridges (circle), gaps in the normally uniform monolayer of nuclei (box), and asynchronous mitoses (middle).

Figure 3.—

Meiotic crossing over in mus309^N2 females. Rates of meiotic crossing over in six intervals spanning distal 2L to proximal 2R are shown. The loci used in mapping are above the graph, placed along the x-axis according to physical distance along the chromosome. The gap between pr and cn indicates the position of the centromere and the pericentric heterochromatin, which is not included in the physical distances shown here. Solid lines show the number of map units (m.u.) per megabase pair (Mb) in each interval for wild-type females (shading) and mus309^N2 females (solid). Dashed lines indicate the mean crossover rate across the entire region assayed for each genotype.

Figure 4.—

Hypersensitivity of mus309 mutants to ionizing radiation. Survival to adulthood of homozygous or heteroallelic mutants, relative to survival of heterozygous controls, is shown for three mutant genotypes for doses of gamma radiation up to 4000 rad. These doses do not have a large effect on survival of wild-type or heterozygous larvae (data not shown). Error bars indicate standard deviation from three independent experiments.

Figure 5.—

Gap repair assay using P{w^a}. (A) Schematic of P{w^a} structure. Green boxes represent exons of the sd gene. P{w^a} is inserted into an intron of this gene. Black rectangles are P-element ends and red rectangles are the w gene. The copia retrotransposon (orange; LTRs indicated by carets) is inserted into an intron of w, decreasing expression such that homozygous females and hemizygous males have apricot-colored eyes, and hemizygous females have yellow eyes (Muller 1932). (B) Transposase-induced excision of P{w^a} leaves a break that has 17-nt 3′ overhangs of P-element sequence. Most repair is believed to occur through SDSA (Kurkulos et al. 1994; Adams et al. 2003; McVey et al. 2004a,b). Completed SDSA can restore the entire P{w^a} (not shown). (C) Completion of SDSA can also involve annealing of the copia LTRs, producing a P{w^a} derivative that retains only one LTR; daughters that inherit this repair product have red eyes. (D and E) The major classes of inaccurate repair. In both cases, the w gene becomes nonfunctional, so daughters that inherit either of these chromosomes will have yellow eyes due to the single copy of P{w^a} inherited from the mother. In D, repair is initiated by SDSA, but is completed by end joining rather than annealing of complementary sequences. In most such cases, synthesis occurs from both ends of the break, as shown here. The extent of synthesis from the right end (dotted double-headed arrow) can be estimated through molecular analysis. (E) In some cases of inaccurate repair, products have a deletion in sequences adjacent to the P{w^a} insertion site. In this example, there has been synthesis from the left end of the break and deletion to the right side (dotted line). Deletions can also be bidirectional. When a deletion extends near or into an exon of sd, as depicted here, the result is a male-lethal allele of sd. Deletions are uncommon in wild-type males, but frequent in mus309 mutants.

Figure 6.—

Gap repair in mus309 mutants. Bars indicate the fraction of repair products that occurred through SDSA with annealing of LTRs (see Figure 5C and materials and methods). For each genotype, the maternally inherited allele is listed above the paternally inherited allele. Bars represent means and lines are standard errors of the mean. Number of vials, progeny, and red and yellow classes are given in supplemental Table S4 at http://www.genetics.org/supplemental/. For statistical analysis, see supplemental Table S5.

Figure 7.—

Germline crossovers in wild-type and mus309 mutant males. Bars show the mean percentage of progeny that were recombinant between st and e, with lines indicating standard error of the mean. Males either were untreated or were exposed to the indicated dose of gamma radiation during larval development. Crossover rates between different mutant genotypes were not significantly different, but at each dose each mutant genotype was significantly different from the wild type (P < 0.0001 for each comparison). See supplemental Table S7 at http://www.genetics.org/supplemental/ for numbers of vials, progeny, and crossovers.

Figure 8.—

Models for DmBlm function in DSB and gap repair. (A) Hypothesized functions for DmBlm in DSBR. (Left) The steps involved in generating a crossover, according to a modified version of the DSBR model of Szostak et al. (1983). (i) Initial processing of the DSB involves resection of the 5′-ends, which generates 3′-ended, single-stranded overhangs. (ii) One overhang invades a homologous duplex, generating a D-loop, and the D-loop is enlarged when the invading strand is extended by repair DNA synthesis. (iii) The displaced strand anneals to the other 3′ overhang. (iv) Additional synthesis extends this end of the break, using the displaced strand as a template, and ligation at both ends leads to a DHJ. (v) The DHJ is resolved by nicking of two strands at each junction. In the example shown here, the two inner (crossing) strands are cut at the left junction, and the two outer (noncrossing) strands are cut at the right junction, giving rise to crossover chromatids. Resolution can also give rise to noncrossover chromatids. In the disruptase model, DmBlm removes the invading strand during or after synthesis, as in the SDSA model. The nascent sequence anneals to the other resected end, resulting a noncrossover repair product. In the dissolvase model, DmBlm migrates the two Holliday junctions toward one another. Decatenation by a type I topoisomerase generates a noncrossover repair product. (B) Hypothesis for the formation of deletions during gap repair. The first diagram illustrates a chromatid from which a transposable element (solid lines) has excised and the ends have been resected. (i) As in DSBR, a resected end invades a homologous template, such as the sister chromatid, and primes new synthesis. Displacement of the D-loop does not extend far enough to allow capture of the second end of the break. (ii) If DmBlm cannot dissociate the invading strand, this strand is cut (open arrowhead), resulting in an enlarged gap. (iii) If the two ends of the enlarged gap are repaired through end joining, as is common during gap repair in Drosophila, the product will lack the transposable element and will be deleted for sequences adjacent to the insertion site. If the left end of the break has invaded the sister chromatid and primed repair synthesis, it may contain some sequences from the left end of the transposable element or may also have a deletion. The fate of the template chromatid is not shown. Cutting of the invading strand may allow another helicase to remove the annealed strand, or it may be removed at the next S phase. A more complete illustration of this model for gap repair is given in supplemental Figure S1 at http://www.genetics.org/supplemental/.