Analysis Of Ubiquitination Of The Small Subunit P50 Of Human DNA Polymerase ?

Human DNA polymerase ?(Pol ?)is a heterotetramer consisting of four subunits: p125,p68,p50 and p12.It is not only the main DNA polymerase in chromosomal replication,but also a participater that is involved in a variety of intracellular repair processes.The high fidelity and processivity of Pol ? make DNA replication accurate and continuous.In other words,p125 catalytic subunit posseses 3'-5' exonuclease activity,which ensures the accurate base pairing during DNA replication.Even rare mismatched bases will be removed by the exonuclease activity,making DNA replication supremely accurate.In addition to its unique role in various DNA repair pathways,Pol ? is also a target of the DNA damage response(DDR).Previous studies showed that p12 subunit is degraded via ubiquitin–proteasome pathway that is mediated by UbcH5c/RNF8(E2/E3)in response to DNA damages that are triggered by UV irradiation or alkylating agents,leading to the conversion of the Pol ? holoenzyme to the heterotrimeric Pol ?3.Pol ?3 exhibits an increased discrimination against mutagenic bypass synthesis and an increased fidelity against insertion of mismatched nucleotides.Besides,we have noticed that p50 subunit might be monoubiquitylated by the same E2/E3 pair in vitro,but a final conclusion has not yet been made.Based on the potential important role of monoubiquitylated p50 subunit in the mediation of exchanging between Pol ? and translesion DNA synthesis(TLS)polymerase in TLS pathways,in this study,we firstly used both UbcH5c/RNF8 and Rad6/Rad18 E2/E3 pairs to confirm the monoubquitination of p50 subunit via the established in vitro ubiquitination analysis system.Also,the other three subunits of Pol ? were analyzed by the same analysis system.The results showed that both E2/E3 pairs can conduct the monoubiquitination of p50,in which the UbcH5c/RNF8 pair appeared a high effciency for the monoubiquitination of p50.However,neither of these pairs could mediate the ubiquitination of p125 and p68 but both of them could mediate the ubiquitination of p12.Next,we examed the performance of the ubiquitination of p50 in UV-treated HeLa cells.Indeed,p50 was found to be monoubiquitinated in response to DNA damage triggered by UV irradiation.Finally,subcellular localization was observed in UV-treated HeLa cells.It was showed that Pol ? is co-localized with RNF8 and Pol ? to DNA damage foci that is induced by UV irradiation.It implys that once UV-induced DNA damage occurs in living cells,p12 is rapidly degraded via ubiquitin–proteasome pathway.In the same time,Pol ?3,RNF8,Pol ?,including other repair-associated proteins such as PCNA,are rapidly recruited to DNA damage foci to participate in TLS.RNF8 may serve as E3 ubiqutin ligase here to mediate the monoubiquitination of p50.According to these results,we proposed a hypothesis to explain the possible role of monoubiquitylation of p50 in polymerase switching during TLS process.This study may provide a theoretical reference for uncovering novel mechanisms as to how Pol ? is exchanged with TLS polymerase in TLS pathway.It also has significant implications in regard to its potential contributions to design novel strategies,making Pol ? as a potential target of small molecule inhibitors,for the prevention and therapy of human diseases such as cancer.