Synthetic lethality of Drosophila in the absence of the MUS81 endonuclease and the DmBlm helicase is associated with elevated apoptosis

Abstract

Mus81-Mms4 (Mus81-Eme1 in some species) is a heterodimeric DNA structure-specific endonuclease that has been implicated in meiotic recombination and processing of damaged replication forks in fungi. We generated and characterized mutations in Drosophila melanogaster mus81 and mms4. Unlike the case in fungi, we did not find any role for MUS81-MMS4 in meiotic crossing over. A possible role for this endonuclease in repairing double-strand breaks that arise during DNA replication is suggested by the finding that mus81 and mms4 mutants are hypersensitive to camptothecin; however, these mutants are not hypersensitive to other agents that generate lesions that slow or block DNA replication. In fungi, mus81, mms4, and eme1 mutations are synthetically lethal with mutations in genes encoding RecQ helicase homologs. Similarly, we found that mutations in Drosophila mus81 and mms4 are synthetically lethal with null mutations in mus309, which encodes the ortholog of the Bloom Syndrome helicase. Synthetic lethality is associated with high levels of apoptosis in proliferating tissues. Lethality and elevated apoptosis were partially suppressed by a mutation in spn-A, which encodes the ortholog of the strand invasion protein Rad51. These findings provide insights into the causes of synthetic lethality.

Figure 1.—

Mutations in mus81 and mms4. Schematics of (A) mus81 and (B) mms4 are shown. Protein-coding sequences are in solid. The region encoding the nuclease domain of MUS81 is stippled. The 16-bp insertion in mus81^Nhe is indicated, and the 3′-end of the adjacent, overlapping gene is shown. In B, the region deleted in mms4^ex1 is denoted with a dashed bar. Both mus81 and mms4 lack introns. The 3′-UTR of each overlaps with the adjacent gene, which is transcribed in the opposite direction.

Figure 2.—

(A–C) Sensitivity to DNA damage in mus81^Nhe. Rates of survival to adulthood were calculated relative to control siblings in the same vial and normalized to ratios in untreated vials. Numbers on the x-axis represent the total quantity of agent added to the food (see materials and methods), except for nitrogen mustard, which indicates the percentage (v/v) of agent in the 250 μl added to ∼8 ml of food. Solid lines, mus81^Nhe; shaded lines, mei-41^29D (positive control to verify activity of the agents). Relative survival for each point is the weighted mean from four to seven independent experiments, each including 7–10 independent vials. Error bars show standard deviation. For each point shown, there is a significant difference between survival of mutant and control siblings (P < 0.05), with the exception of mus81^Nhe at the lower hydroxyurea dose (P = 0.1221).

Figure 3.—

Apoptosis in proliferating tissues. (A) Wing imaginal discs from mature third instar larvae, stained with an antibody that recognized cells undergoing apoptosis. Representative discs from four different genotypes are shown. The mus309^N1 null allele was used in all cases. (B) Quantification of apoptosis in wing discs from larvae of various genotypes. Genotypes are given below the graph, with an “X” indicating a null mutation in the corresponding gene. Each bar represented the mean number of apoptotic cells (n = 10, 5, 15, 12, 12, 11, 13, and 8, left to right). Error bars are standard error of the mean.

Figure 4.—

Conceptual model for roles of SPN-A, DmBlm, and MUS81 in repair of spontaneous lesions. (A) Type I lesions are normally processed by SPN-A into an intermediate that is further processed by DmBlm. Our data do not reveal any evidence for a role of MUS81 in repairing this type of lesion. (B) Type II lesions are processed by either of two pathways, one requiring MUS81 and the other requiring DmBlm. Our data do not reveal a role for SPN-A in repairing this type of lesion.