Study On The Role And Mechanism Of RPA Chaperone Rtt105 In DNA Double-strand Break Repair

Homologous recombination and non-homologous end joining are two main approaches to repair DNA double-strand breaks(DSBs).The repair process of homologous recombination(HR)is more complex and involves more proteins.Among these proteins,RPA is one of the key proteins in homologous recombination,which can bind and protect single strand DNA(ssDNA),mediate the activation of cell cycle checkpoint,and participate in DNA replication,transcription,HR,DNA damage repair and other DNA transaction processes.As the chaperone of RPA,Rtt105 is involved in DNA replication.In order to understand the function of Rtt105 as an RPA chaperone on other cellular processes,we explored its role and mechanism in HR repair,and obtained the following conclusions:1.Rtt105 is involved in DNA damage repair.We found that RTT105 knockout cells were sensitive to multiple DNA damage drugs based on drug sensitivity assays.After a brief drug treatment,the survival rate of RTT105 knockout cells decreased significantly as compared to wild-type cells,indicating Rtt105 play an important role in the DNA damage response.In addition,we found that in the RTT105 knockout cells,the Rad52-YFP focus(foci)is significantly higher than that of wild-type cells,indicating that RTT105 knockout cells accumulated spontaneous DNA damage.By microscopy analysis,we found that Rtt105 promoted the recovery from DNA damage.This is further confirmed by pulsed field gel electrophoresis(PFGE)experiment.2.Rtt105 promotes DSB repair by HR.We detected the efficiency of DSB repair by HR,and found that the efficiency of HR repair decreased significantly in RTT105 knockout cells,indicating that Rtt105 promoted HR repair.This conclusion is confirmed by gene targeting assay.Further,by chromatin immunoprecipitation(ChIP)assay,we found that Rtt105 was recruited to the end of DSBs,in a manner dependent on end resection but not checkpoint signaling.As a sequence,RTT105 knockout cells showed increased loss of yeast artificial chromosomes(YAC),suggesting a role of Rtt105 in maintaining genomic stability.3.Rtt105 regulates the nuclear localization of Rfa1.We found that the phosphorylation of Rad53 induced by DSBs was normal in RTT105 knockout mutants,indicating that Rtt105 does not affect the activation of DNA damage checkpoint.Using fluorescence microscope,we observed that in wild type cells,Rfa1 fully distributes in the nucleus,while in RTT105 knockout cell,Rfa1 distributes in both the nucleus and cytoplasm,indicating that Rtt105 could regulate the nuclear localization of Rfa1.Further studies showed that in RTT105 knockout cells,fusion of nuclear localization signal sequence at the N-terminal of Rfa1 could fully restore the nuclear localization of Rfa1.The interaction between RTT105 two-point mutation(rtt105-E171AL172A)and Rfa1 was greatly reduced.We noted that two-point mutation did not affect the nuclear localization of Rfa1,but it showed defects in homologous recombination,suggesting that the role of Rtt105 in regulating the nuclear localization of Rfa1 is not relying on its interaction with Rfa1.It also indicates that the effect of Rtt105 on homologous recombination is not mainly dependent on its effection on RPA nuclear localization,suggesting that Rfa1-Rtt105 interaction may have other functions.4.Rtt105 promoted the recruitment of Rfa1 and Rad51 at the DNA double strand break ends.By ChIP experiments,we found that Rtt105 promoted the binding of Rfa1 and Rad51 at DSB ends.The binding amount of Rfa1 at DSBs in two-point mutant cells was lower than in wild type cells,indicating that the interaction between Rtt105 and Rfa1 helps Rfa1 bind at DSB ends.Fusion of a nuclear localization signal(NLS)at the N-terminus of Rfa1 restored its nuclear localization,partially resue the Rfa1 loading defect in RTT105 knockout cells,indicating that the function of Rtt105 promoting Rfa1 nuclear localization could help Rfa1 bind on the DNA double strand break ends..5.The C-terminal of Rtt105 promotes DSBs repair.By fluorescence microscopy analysis,we found that the region between residue 155 and 175 of Rtt105 is critical for proper Rfa1 nuclear localization.In addition,we found that this region was also critical for DNA damage repair and repair by HRThe above results show that Rtt105 regulates the function of RPA by controlling the behavior of RPA,including controlling the nuclear localization of RPA and helping RPA to bind at DSB ends,thus regulating the repair by HR.