Temporal Analysis of Meiotic DNA Double-Strand Break Formation and Repair in Drosophila Females

Abstract

Using an antibody against the phosphorylated form of His2Av (γ-His2Av), we have described the time course for the series of events leading from the formation of a double-strand break (DSB) to a crossover in Drosophila female meiotic prophase. MEI-P22 is required for DSB formation and localizes to chromosomes prior to γ-His2Av foci. Drosophila females, however, are among the group of organisms where synaptonemal complex (SC) formation is not dependent on DSBs. In the absence of two SC proteins, C(3)G and C(2)M, the number of DSBs in oocytes is significantly reduced. This is consistent with the appearance of SC protein staining prior to γ-His2Av foci. However, SC formation is incomplete or absent in the neighboring nurse cells, and γ-His2Av foci appear with the same kinetics as in oocytes and do not depend on SC proteins. Thus, competence for DSB formation in nurse cells occurs with a specific timing that is independent of the SC, whereas in the oocytes, some SC proteins may have a regulatory role to counteract the effects of a negative regulator of DSB formation. The SC is not sufficient for DSB formation, however, since DSBs were absent from the heterochromatin even though SC formation occurs in these regions. All γ-His2Av foci disappear before the end of prophase, presumably as repair is completed and crossovers are formed. However, oocytes in early prophase exhibit a slower response to X-ray–induced DSBs compared to those in the late pachytene stage. Assuming all DSBs appear as γ-His2Av foci, there is at least a 3:1 ratio of noncrossover to crossover products. From a comparison of the frequency of γ-His2Av foci and crossovers, it appears that Drosophila females have only a weak mechanism to ensure a crossover in the presence of a low number of DSBs.

Evidence based on studies of the mus309 mutant, deficient in DNA double-strand break repair, that meiotic crossing over in Drosophila melanogaster is a two-phase process

“…These broods are known to represent oocytes which were in the premeiotic DNA synthesis stage during the treatment (Grell and Chandley 1965;Grell 1973 andMehrotra andMcKim 2006). However, this period is not coincidental with the occurrence of crossing over, but represents the time of some precondition of crossing over as was first shown by Portin and Suormala (1973).…”

“…This is in accordance with the view that the DNA synthesis associated with crossing over is involved in the repair of DSBs (see Introduction). In addition the recent molecular genetic analyses also suggest that crossing over occurs later in meiosis, most likely during the pachytene stage of the prophase (Mehrotra andMcKim 2006 and.…”

“…According to Mehrotra and McKim (2006) there are 20-24 c-His2Av sensitive DSBs in the late pachytene oocytes of the wild type D. melanogaster (c-His2Av is a phosphorylated variant of histone 2A, being an excellent cytological marker for meiotic DSB formation). In contrast, extensive genetic studies have shown that there are only approximately six crossovers per Drosophila female meiosis (reviewed in McKim et al 2002).…”

Meiosis as an Evolutionary Adaptation for DNA Repair

“…In mouse meiosis there are > 10-fold more DSBs than CO recombinants (Moens et al, 2002), suggesting that most DSBs are repaired by NCO recombination. In D. melanogaster there is at least a 3:1 ratio of NCOs to COs (Mehrotra & McKim, 2006). These observations indicate that the majority of recombination events are NCOs.…”

Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress

“…To investigate this, we labeled cells with an antibody against the phosphorylated form of histone H2AV (␥-H2AV). H2AV is phosphorylated on serine 137 by the checkpoint kinase ATM at sites of DNA damage, which can be seen as nuclear repair foci after labeling with anti-␥-H2AV antibodies (Madigan et al 2002;Mehrotra and McKim 2006). ATM is also the proximal checkpoint kinase that initiates the apoptotic pathway.…”