Dynamic RPA Acetylation And Deacetylation Promote Accurate DNA Replication And Repair

The errors accumulated during DNA replication and repair can lead to genome instability and tumorigenesis.In cancer cells,dysfunctions of different DNA repair or replication proteins cause complex genome rearrangement and mutation events.Therefore,it is crucial to restrain mutations and the usage of low-fidelity repair to prevent tumorigenesis and other diseases.The single-strand DNA(Single-stranded DNA,ss DNA)binding protein RPA(Replication protein A,RPA)plays a critical role in promoting high-fidelity DNA replication and repair.As the major eukaryotic ss DNA binding protein,RPA has a high affinity with ss DNA and acts as the first responder for ss DNA.RPA is essential for the fundamental nuclear processes.Dysfunction of RPA can give rise to mutation,genome instability,chromosome breakage,and carcinogenesis.RPA is known to be regulated by several different types of post-translational modifications,including acetylation.However,how RPA is precisely regulated to ensure high-fidelity replication and repair is still unknown.In this study,we focused on the functions of RPA acetylation in maintaining the fidelity of DNA replication and repair and genome stability and obtained the following results:1.By immunoprecipitation,we found that RPA1 acetylation was stimulated by the DNA damage induced by MMS.Using in vitro and in vivo assays,we demonstrated that the RPA complex was primarily acetylated by the acetyltransferase Nu A4 on Rfa1.Simultaneous mutation of K259,K427,K463,and K494 to arginine(4KR)significantly impaired RPA acetylation and caused sensitivity specific to the DNA damaging agent phleomycin or zeocin.While simultaneous mutation of the four residues to glutamine(4KQ)led to hypersensitivity to multiple DNA damaing agents,including phleomycin,MMS,camptothecin and zeocin.These results suggested that K259,K427,K463,and K494 were the major acetylation sites on RPA and dynamic acetylation and deacetylation of RPA promoted DNA damage response and repair.2.Using multiple DNA double-strand break(DSB)repair reporter systems to explore the roles of RPA acetylation,we observed that the 4KQ mutation impaired DSB repair by gene conversion(GC)or break-induced replication(BIR)while increasing the repair by alternative end joining(alt-EJ).In addition,it also decreased the repair by single-strand annealing(SSA).Notably,the 4KR mutation caused reduced gene conversion and increased alt-EJ and SSA repair.These results suggested that proper RPA acetylation and deacetylation promoted high-fidelity repair pathways while suppressing the error-prone repair mechanisms.3.Further,we found that mimicking persistent RPA acetylation(4KQ)increased spontaneous mutation rates by 5-fold.Meanwhile,we found that both the4 KQ and 4KR mutant cells carry the characteristic mutations with the signature of micro-homology-mediated large deletions or insertions.In addition,using a yeast artificial chromosome(YAC)system,we found that the frequency of chromosome loss significantly increased in the 4KQ and 4KR mutant.These results suggest that dynamic RPA acetylation and deacetylation promoted high-fidelity replication while suppressing mutations and chromosome loss.4.For the sake of analysing the mechanism of RPA acetylation,we conducted EMSA,single-molecular magnetic tweezers and streptavidin pull-down assays and we provided in vitro evidence that the 4KR mutant RPA protein had a reduced ss DNA binding ability,while the defect was more severe for the 4KQ mutant RPA.Using the chromatin Immunoprecipitation(Ch IP)assay,we showed that the loading of RPA,Rad51 and RAD52 at DSB ends was significantly decreased.These results indicated that dynamic RPA acetylation and deacetylation regulated the binding of RPA and ss DNA in vitro and in vivo.5.We analyzed the subcellular distribution of RPA in the WT、4KQ or 4KR cells using fluorescent microscope.We found that RPA nuclear localization was slightly changed in the 4KR mutant cells and became abnormal in the 4KQ mutant cells,suggesting that proper RPA acetylation is required for RPA nuclear localization.Interestingly,we noted that the 4KQ mutant RPA exhibited an enhanced binding ability to Rtt105 and Kap95,two proteins involved in RPA nuclear import.Furthermore,we found that Rtt105 and 4KQ functioned epistatically to promote RPA nuclear localization,HR repair and the response to DNA damage.6.Similarly,simultaneous mutation of K259,K458 and K489 to arginine(3KR)in human RPA significantly impaired RPA acetylation and its binding ability to ss DNA,suggesting that the three residues in human RPA are acetylated.Using multiple DSB reporter assays,we observed that mimicking constitutive RPA acetylation(3KQ)led to attenuated repair by HR or BIR and slightly increased repair by alt-EJ and SSA.Blocking RPA acetylation(3KR)also compromised the repair by HR.These results suggested that timely RPA acetylation and deacetylation promoted high-fidelity replication and repair while suppressing the deleterious repair pathways in human cells.In summary,by a combination of in vivo and in vitro assays,we provided evidence that dynamic RPA acetylation and deacetylation promoted accurate DNA replication and repair,while restraining mutations and the usage of low-fidelity repair mechanisms.We showed that this regulatory mechanism was likely conserved across species.This study revealed a novel function of RPA acetylation in preserving genome stability and provided an insight into how the dynamic binding of RPA to ss DNA was regulated and how genome integrity was maintained.