Functional Studies Of Drosophila PTIP In DNA Repair

Various kinds of DNA lesions have been considered as one of the major causes for cell damage and cell death.In order to survive,Living organisms ranging from prokaryotes to eukaryotes have evolved effective yet complicated DNA damage response(DDR)pathways to recognize and to repair these DNA damages.Therefore,it is not surprising that DDR defects are closely linked to many human diseases including cancer.Taken the single most severe form of DNA damages double strand breaks(DSB)for example,in higher organisms,multiple factors are known to be part of delicate DDR pathway,many of which are phosphorylated by ATM/ATR kinases and form a DDR cascade.Eventually DSB are thought to be repaired by distinct means,namely homologous repair(HR)and non-homologous end-joining(NHEJ),with high fidelity of the former one and error-prone fashion of later one.Choice making between HR and NHEJ are crucial for efficient DSB repair.On one hand it could be regulated by cell cycle checkpoints,such that NHEJ for G1 phase when DSB frequency is low and HR for S/G2 with much higher DSB occurrences.On the other hand,BRCT-domain containing proteins,most notably BRAC1,bearing a BRCT pair at its C-terminal,whose mutations are a high risk factor for Breast cancer.The BRCT pair has been suggested as ATM/ATR substrates recognition module in their Ser/Thr phosphorylated forms.This study focused on another BRCT protein called Pax IP1,or PTIP,and its function in DSB repair.Like BRAC1 in mammalian systems,PTIP serves as a recognition module for phosphorylated ATM/ATR substrates.Several studies also indicate that PTIP is a major regulator in NHEJ/HR choice making and loss of PTIP may induce severe genome instability.Due to its highly pleiotropic nature,PTIP also functions in transcription regulation and DNA recombination.PTIP is highly conserved in metazoan ranging from fruit fly to human.Domain structures of Fly PTIP is very similar to that of mammalian counterparts,all composed by three BRCT pairs and an extensive Q-rich region following the N-terminal pair.However little is known if and how PTIP could have a role in DDR,which left us the major scientific question to answer in the present study.We used a newly constructed Ptip mutant allele in addition to two previously published ones to look closely on how loss of function of Ptip would have an effect on development,growth,cell apoptosis and vulnerability to DNA damage.We found a compelling less number of third instar zygoticly heterozygotic Ptip mutant larvae,suggesting that Ptip is essetial for larval development.Further more,mutant wing imaginal discs were absent,smaller and deformed,consistent with our previous observation in our lab.By immunostain we present evidence that loss of Ptip play profound roles in cell growth,by increased cell apoptosis and decreased proliferations.The dramatic augment of ?H2AV signal,barely seen in normal discs,indicating PTIP may be essential for cell growth by the maintenance of proper DDR function.Through the Qpcr experiment,we found that the expression of the main target gene of NHEJ/HR in the third instar zygoticly heterozygotic Ptip mutant larvae was reduced,indicating the necessary role of Ptip in DSB repair.Finally,we showed that Ptip deficient gut cells were bearing increased vulnerability to DNA damaging agents.At the same time,we observed the increase of ?H2AV signal in Drosophilia Kc cells after Ptip Knockdown(ctrl Kc cells had almost no ?H2AV signal)through the experimental method of RNAi,and the increase of ?H2AV signal was more significant after the treatment with external DSB damaging agent Dox.We conclude that PTIP may regulate DDR in a conserved fashion in Drosophila.We are currently carrying out an in vivo experiment examining the possible role of Ptip in NHEJ/HR repair choice making.These results pave the way to further elucidation of detailed mechanism underlying the role of PTIP in DDR.For example,it has been shown that PTIP helps class-switch recombination in mouse B cell development by its interacting Histone methyltransferase protein complex and the H3K4 trimethylation in recombination sites.The model we created in Drosophila system would be of great help in deciphering links between Histone modification and DDR and repair choice making.