Functional Analysis Of DNA Repair Related Polymerases In Deinococcus Radiodurans

Deinococcus radioduransis is highly resistant to extraordinary doses of ionizing radiation,desiccation,ultraviolet,and a variety of chemical mutagens,which serves as an ideal model organism for elucidating DNA repair system.Since almost all the DNA repair pathways require DNA polymerases,the investigation of DNA repair-related polymerases in D.radiodurans will provide further insight into DNA damage repair mechanisms.The genome of D.radiodurans encodes three DNA polymerases:Pol?,Pol? and PolX.Pol? is the primary DNA replication polymerase,while Pol? and PolX are mainly participated in DNA repair.DrPol1 was found to be essential for the specific Extension synthesis-dependent strand annealing(ESDSA)repair pathway in D.radiodurans.DrPolX has been reported to participate in Double-strand break repair(DSBR)and Base excision repair(BER)pathways.Here we systematically investigated the biological function of DrPol1 and DrPolX both in vitro and in vivo.DrPol1,a DNA polymerase of the A family,contains two relativity independent domains:C terminal Klenow fragment(KlenDr)and N terminal nuclease domain(DrPol1-NTD).In this study,we purified DrPol1 and its separated domains respectively and characterized their biochemical features in DNA repair processes:KlenDr shows high polymerase fidelity despite the lacking of 3?-5?exonuclease proofreading activity.Its nucleotide incorporation accuracy is related to the flexible linker between the N-helix and O-helix in the finger domain of KlenDr.Different from the Klenow fragment of E.coli Pol1,KlenDr prefers gapped DNA rather than Primer-Template substrates.DrPol1-NTD exhibits similar flap endonuclease(FEN)activity with h FEN1 and its homolog in E.coli but with over 100-fold higher gap endonuclease(GEN)activity than them.DrPol1-NTD could also cleave the Holliday Junction structures efficiently.The DrPol1-NTD deficient strain shows increased sensitivity to UV and gamma-ray radiation in vivo.The polymerase domain contributed to the majority DNA binding affinity of DrPol1 and increased the endonuclease activity of DrPol1 to complex DNA structures.DrPol1 first extended the P-T substrates then performed exonuclease activity in vitro.DrPolX is a member of X family DNA polymerase,which contains polymerase(PolX-core)and Polymerase and Histidinol Phosphatase(PHP)domain.However,these two domains are tightly associated and could only perform biochemical activities with the full-length protein.PolX-core responsible for the DNA binding affinity of DrPolX,whereas the PHP domain helped the recognition of specific DNA structures.Apart from the well-reported 3?-5?exonuclease activity,DrPolX is also endowed with AP endonuclease activity,which is important in the BER repair pathway.The DrPolX^H334A mutation in DrPolX abolished more than 90%of the AP endonuclease activity,but retained the 3?-5?exonuclease activity,while the DrPolX^D339A mutation in DrPolX displayed an opposite change in these two activities.DrPolX contains an“AEE”DNA polymerase catalytic motif,which is different from the conserved“DDD”motif among other X family polymerases.However,we did not observe the polymerase activity of DrPolX in vitro after attempting various reaction conditions.Besides,knocking out of drpol X did not significantly decrease the resistance of D.radiodurans to MMS and UV radiation,suggesting that the biochemical activity of DrPolX in the BER repair pathway might be compensated by other isoenzymes in vivo.In summary,we systematically characterized the DNA repair-related biochemical properties of DrPol1 and DrPolX,explored the collaboration between their nuclease and polymerase domains,and confirmed the biological function of DrPol1 and DrPolX for the extreme resistance of D.radioduransin vivo.These results contributed to a better understanding of DNA repair-related polymerases and provide further information to the DNA damage repair system.