Dm brca2 in DNA Repair and Checkpoints Figure 4. brca2 Is Not Required for the Checkpoints That Respond to Irradiation Damage Climbing third instar larvae were irradiated and then dissected prior to fixation. Error bars represent the standard error from a total of 6–10 samples from 2–3 separate experiments. Representative images can be found in Figure S1. (A) Larvae were fixed 1 hour after a 4,000 Rad IR exposure and stained for the mitotic marker phospho-histone H3. n ¼ 6. (B) Larvae were fixed 4 hours after a 4,000 Rad IR exposure and stained for activated Caspase-3. n ¼ 6. (C) Larvae were fixed 1 hour after a 500 Rad IR exposure and stained for the mitotic marker phospho-histone H3. n ¼ 11. (D) Larvae were inverted 1.5 hours after a 4,000 Rad IR exposure and incubated in Schneider’s media with BrdU for 30 minutes. n ¼ 7–10. doi:10.1371/journal.pgen.0040031.g004 and mediator proteins [43]. However, each checkpoint has speciﬁc genetic requirements for signal transduction. There- fore, we thought it possible to gain a better understanding of where in the meiotic recombination checkpoint pathway Brca2 acts by examining whether brca2 was required for other Drosophila checkpoints. We used the well-established assays in the imaginal discs and brain of the Drosophila larvae to monitor the checkpoints that respond to irradiation in animals for brca2. Irradiated imaginal discs exhibit a G2 cell cycle arrest within 1 hour of irradiation and induce apoptosis within 4 hours post-irradiation [44,45]. The induction of apoptosis requires chk2 and p53, but not mei-41 or grp/chk1 [46]. Cell cycle arrest after high dose IR (4,000 Rads) requires mei-41, to lesser extents chk2 and grp, but not atm [38,44,46]. Cell cycle arrest after low dose IR (500 Rads) requires mei-41 as well as atm [47]. A mei-41-dependent decrease in replication, or an intra-S phase checkpoint, is seen within 2 hours post-irradiation in the larval brain lobes [48]. We did not see any requirement for brca2 in the checkpoints that respond to irradiation (Figures 4 and S1). After a 4,000 Rads IR exposure, brca2 mutants demonstrated efﬁcient G2 arrest (Figure 4A) and induction of apoptosis (Figure 4B). Cell cycle arrest after a 500 Rad IR exposure was also not compromised in brca2 mutants (Figure 4C). We did not see any difference between brca2 mutants and wild-type larvae in the reduction of 5-bromo-2-deoxyuridine (BrdU) staining after a 4,000 Rad exposure (Figure 4D). However, unlike the cell cycle and apoptosis assays in which the difference before and after irradiation is dramatic, the intra- S checkpoint effect is a reduction in replication levels by slightly less than half (Figure 4D). Therefore we do not expect to be able to detect partial requirements for genes involved in the intra-S phase checkpoint. Collectively, the irradiation checkpoints we tested required atm, mei-41/atr, chk1, and chk2 and did not require brca2, demonstrating that the activation of the core checkpoint transducers in response to irradiation in Drosophila does not require brca2. In addition to testing for a requirement for brca2 in the irradiation checkpoints we also examined hus1 mutants. Hus1 is a member of the 9-1-1 complex required for activation of the Drosophila meiotic recombination checkpoint and has been shown to be involved in S-phase checkpoints in mammals [36,49]. Similar to brca2, hus1 is not required for the cell cycle arrest or apoptotic induction after high dose IR in Drosophila [36]. In this present study we found that hus1 is also not required for the G2/M checkpoint after low dose IR or the intra-S checkpoint (Figure 4C and 4D). While we PLoS Genetics | www.plosgenetics.org 0007 February 2008 | Volume 4 | Issue 2 | e31