On the roles of Polq, Parp, and Rif1 in DNA Repair
Krishnamurthy, Manan
2021
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Polq and Parp Abstract: Polymerase theta (POLθ) is important for the repair of DNA double strand breaks (DSBs) by theta mediated end joining (TMEJ) and for interstrand crosslink (ICL) repair in Drosophila melanogaster. However, the upstream regulation that precedes POLθ recruitment and function is poorly understood. In vitro and ex vivo studies in mammalian cells have implicated PARP-1 as necessary ... read morefor the initiation of TMEJ, albeit by an unknown mechanism. Given the high levels of functional conservation of POLθ and structural conservation of PARP between Drosophila and humans, the Drosophila model system provides an excellent opportunity to probe this relationship in vivo. In order to do so, bleomycin sensitivity assays and hatching assays were conducted following RNA interference (RNAi) mediated knockdown of PARP. Next, PARP catalytic inhibition by veliparib dihydrochloride was used to test the hypothesis that PARylation is important for the recruitment of POLθ to DSBs or ICLs. Specifically, comparative analysis was employed following bleomycin and nitrogen mustard sensitivity assays simultaneously treated with veliparib dihydrochloride. Since previous studies from the lab have shown that polq null mutants are only sensitive to ionizing radiation (IR) in an HR deficient rad51 null background, these assays were conducted in polq null, rad51 null, and polq null-rad51 null mutants. RNAi-mediated knockdown of flies was inconclusive. PARP inhibition did not confer increased sensitivity to bleomycin or nitrogen mustard in the polq null or rad51 null mutants, but conferred a slight increase in the polq null-rad51 null double mutants. Overall, this suggests that PARP catalytic activity does not serve a role in the initiation of TMEJ or ICL repair in flies. Interestingly, polq null mutants were found to be sensitive to bleomycin despite their lack of sensitivity to IR suggesting that different repair mechanisms are required to repair IR and bleomycin induced DSBs. Additionally, we have also endogenously tagged Polq using a scarless CRISPR knock-in as a means of probing POLθ recruitment to DNA damage in future studies.Rif1 Abstract: Rap1 interacting factor 1 (Rif1) is a structurally well-conserved protein in eukaryotes that plays an important role in pathway choice during the repair of DNA double strand breaks (DSBs). In human cells, Rif1 directly interacts with phosphorylated p53 binding protein (53BP1) to inhibit BRCA1 mediated resection of DSBs during G1, and hence promotes repair via non-homologous end joining. In contrast, Saccharomyces cerevisiae Rif1 was shown to promote resection and homology-directed repair of DSBs in both G1 and G2. Given the opposing repair functions of Rif1 in evolutionary distant mammalian and yeast cells, we have explored the DSB repair role of Rif1 in vivo in phylogenetic intermediate Drosophila melanogaster. Hatching assays were conducted to determine whether rif1 mutant fly stocks exhibit any DSB repair defect during embryonic development or larval development. No such defect was observed. Next, in order to probe whether Rif1 influences repair pathway choice, as is seen in mammals and yeast, three site-specific DSB assays were conducted. These assays allow for the determination of repair pathway choice employed to repair site-specific DSBs in the male germ-line. rif1 null mutants exhibited defects in repair in each of these assays, collectively suggesting that that Drosophila Rif1 plays a role in the inhibition of uncontrollable extensive resection at DSBs.
Advisor: Professor Mitch McVeyread less - ID:
- xg94j434s
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