Drosophila ATR in double-strand break repair

Abstract

The ability of a cell to sense and respond to DNA damage is essential for genome stability. An important aspect of the response is arrest of the cell cycle, presumably to allow time for repair. Ataxia telangiectasia mutated (ATM) and ATR are essential for such cell-cycle control, but some observations suggest that they also play a direct role in DNA repair. The Drosophila ortholog of ATR, MEI-41, mediates the DNA damage-dependent G2-M checkpoint. We examined the role of MEI-41 in repair of double-strand breaks (DSBs) induced by P-element excision. We found that mei-41 mutants are defective in completing the later steps of homologous recombination repair, but have no defects in end-joining repair. We hypothesized that these repair defects are the result of loss of checkpoint control. To test this, we genetically reduced mitotic cyclin levels and also examined repair in grp (DmChk1) and lok (DmChk2) mutants. Our results suggest that a significant component of the repair defects is due to loss of MEI-41-dependent cell cycle regulation. However, this does not account for all of the defects we observed. We propose a novel role for MEI-41 in DSB repair, independent of the Chk1/Chk2-mediated checkpoint response.

Figure 1.—

Lethality caused by transposase-induced DSBs. (A) Lethality using chromosome 2 transposase and a P{w^a} element. (B) Lethality using chromosome 3 transposase and three P elements (P{w^a} and two at the sn locus). DSBs occur in the somatic tissue and germline of the male progeny of the indicated zygotic genotype that have both the P{w^a} element and transposase (A) or sn^w and P{w^a} elements and transposase (B; see materials and methods for details). The percentage expected is determined by the number of males of the indicated genotype with transposase relative to brothers without transposase. Bars represent means; error bars indicate standard deviation from three independent experiments (wild-type, mei-41, and grp lok mutants) or range from two experiments (other genotypes).

Figure 2.—

Model for repair of a gap through SDSA. Processing of a double-strand gap (a) begins with resection of the ends to leave 3′-ended single-stranded overhangs (b). The resected structure can then enter one of two pathways. The ends can join through a process that does not require extensive complementary ends (c) or one or both of these resected ends invade a homologous template (d) and prime repair synthesis (e). The nascent strand is dissociated, searching for a complementary single-stranded DNA to anneal to f. This structure resembles that of a resected product (b). Consequently, the nascent strand can reinvade a homologous sequence (g) or undergo end joining (i). Repair synthesis is not processive, so multiple rounds of synthesis, dissociation, and reinvasion (e–g) are required for repair across the gap (McVey et al. 2004a). Synthesis across the entire gap allows annealing of complementary single-stranded DNA, resulting in restoration of sequences lost when the gap was originally made (h). In the absence of SPN-A (DmRad51), repair occurs through an end-joining pathway (c). In the absence of MEI-41 (h), a majority of repair products that attempt the annealing/ligation steps are lost, resulting in a reduction of complete SDSA.

Figure 3.—

Percentage of SDSA repair events in checkpoint mutants. Bars represent average percentage of complete SDSA repair (red-eyed progeny) out of all repair products (red- and yellow-eyed progeny) from independent experiments of each indicated genotype. Error bars represent standard deviations from three or four independent experiments in wild type, mei-41, and grp lok mutants and ranges from two independent experiments in lok and grp single mutants. Statistical significance (P < 0.05) was determined from the weighted averages and weighted standard deviations of each genotype. Each independent experiment was an observation of a range of 63–387 individual repair events. See supplemental Table 1 at http://www.genetics.org/supplemental/ for total numbers scored.

Figure 4.—

Synthesis tract lengths in wild-type and mei-41 mutants. Repair synthesis from the right end of the DSB was analyzed for incomplete SDSA repair events (83 independent events from wild-type male flies and 103 from mei-41 mutants). Each bar represents the percentage of repair products with a synthesis tract at least as long as the indicated distance.

Figure 5.—

Checkpoint defects in grp and lok mutants. (A) Schematic of the genomic architecture of grp is shown. Each box represents an exon; solid regions designate protein-coding regions (the four alternative first exons, which are noncoding, are not shown). grp²⁰⁹ was generated by excision of EP587 (triangle) to generate a deletion that removes the first two coding exons (brace). grp^Z5170 has a C-to-T transition that changes a conserved proline at residue 189 in the kinase domain to leucine (asterisk; see materials and methods for details). (B) The lok³⁰ allele was generated by excision of EY15840 (triangle), generating a deletion of the 5′-UTR and the first two coding exons (brace). (C) DNA damage checkpoint defects in mutants. Third instar larvae of the indicated genotype were unirradiated (top) or irradiated with either 500 rad (middle) or 4000 rad (bottom) of γ-rays. Imaginal discs were dissected and fixed 1 hr after irradiation. Mitotic cells are revealed by staining with an antibody to phosphorylated histone H3.

Figure 6.—

Model for MEI-41 in repair of DSBs. We propose that MEI-41 is involved in the later steps of SDSA in repairing a single DSB (after strand invasion and repair synthesis). Our genetic experiments indicate that in addition to the GRP/LOK-dependent checkpoint response, MEI-41 is also involved in repairing DSBs independent of this checkpoint. The presence of MEI-41 in both of these pathways ensures complete repair through SDSA.