The Molecular Mechanism Of Replication Protein A(RPA) Deposition Onto Single-stranded DNA(ss DNA)and Its Role In Genome Stability

Replication stress is the major source of endogenous DNA damage.If replication stress cannot be effectively resolved,the stalled replication fork may collapse to produce DNA double-strand breaks,which seriously threaten the integrity of the genome and lead to accumulation of mutations and cause cell aging or canceration.Unlike normal cells,tumor cells carrying active oncogenes or defective tumor suppressor genes are usually accompanied by high levels of replication stress.This intrinsic feature leads to an increased dependence of tumor cells on the response to replication stress,making this signaling pathway an important target for the treatment of tumors.Regardless of whether the replication fork is stalled,collapsed,or the end-cutting process following DNA breaks,a long single-stranded DNA(ss DNA)coated with RPA(replication protein A)is generated.This ubiquitous intermediate product is an important platform for recruiting and exchanging factors or protein complex(es)involved in replication stress response or DNA damage repair,and it acts as a key structure for DNA damage signal sensing and transmission.RPA-ss DNA mediated signal transduction and replacement of RPA on ss DNA were well understood,but the regulation of RPA binding to ss DNA is still at the beginning of investigation.In order to gain a better understanding of the regulatory mechanism of RPA binding to ss DNA and identify new targets for the treatment of tumors,we aimed to characterized factors that are able to interact with free RPA and actively load RPA on ss DNA.We found the lamina-associated protein LAP2α directly interacted with RPA via SILAC,co-IP,and in vitro pull-down experiments.Next,through EMSA and i POND,high content screening(HCS),laser micro-irradiation and other experiments,we revealed that LAP2α could help RPA deposit onto ss DNA in vitro and in vivo.Mutational analysis indicated that Arg 86 and Arg 88 of LAP2α are critically required for the interaction of LAP2α with RPA,and the charge reversal mutant(R86E/R88E)dramatically compromised LAP2α-promoted RPA loading.Current understanding of LAP2α in tumorigenesis is mainly due to its involvement in LMNA and p Rb-mediated cell cycle control.The role of other related basic cell physiological activities,such as DNA replication,replication stress or DNA damage response,has not been fully studied and explored.Since RPA-ss DNA plays an important role in maintaining genome stability,we next investigated whether LAP2α-mediated RPA deposition was also involved in this process.Through DNA fiber assay,comet assay,HR reporter,MTS,and ATR kinase activity detection experiments,we found that in the LAP2α deficient cells,fork progression on damaged DNA was slowed down,the ability to repair DNA damage was weakened,the ATR signal pathway activation was blocked,and the drug sensitivity was increased.Then,through the Lac I-Lac O system and laser micro-irradiation experiments,we further proved that LAP2α could be recruited to the blocked replication fork or DNA damage sites,and the recruitment was dependent on the poly ADP-ribose polymerase 1(PARP1)but independent of its enzymatic activity.Collectively,we showed that LAP2α is essentially required for genome stability via facilitating fork progression,activating ATR and promoting homologous recombination of DSBs,and it plays a critical role for cells to cope with replication-associated DNA damage.Next,through immunohistochemistry and bioinformatics analysis with oncomine database we found that LAP2α was highly expressed in breast cancer,and its high expression was positively correlated with the poor outcome of breast cancer patients.We demonstrated that LAP2α deletion severely suppressed the growth of breast tumor via mouse breast cancer animal model.Similar to the observations from tumor cells,the level of γH2AX was elevated,indicating that LAP2α deficiency results in an accumulation of DNA damage.We proposed that LAP2α knockout induced tumor growth retardation could be,at least,attributed to an accumulation of replication-associated DNA damage in mammary tumor cells.Moreover,we found that breast cancer cell lines with different molecular types were more sensitive to chemotherapy drugs when LAP2α was knocked down.In summary,we report a LAP2 isoform-specific RPA loading mechanism that regulates the responding of cells to replication stress and DNA damage.By using a combination of biochemical and single cell imaging approaches,we provided an insight of LAP2α-dependent RPA-ss DNA formation and revealed that it is necessary for ATR activation and repair of DSBs.We also revealed that LAP2α is critically involved in breast carcinogenesis,and targeting LAP2α could enhance the efficacy of chemotherapeutics through impairing RPA loading thus exacerbating replication stress and DNA damage induced cell death.