Study On Lesion-bypass Mechanisms Of A Novel Archaeal B-family DNA Polymerase

DNA damage repair is vital for maintaining genomic stability.Previous studies have shown that various endogenous and exogenous DNA damage factors can cause different types of DNA damage to genomes in the cell.To repair these damages,organisms of the three domains of life(Bacteria,Archaea,and Eukarya)have evolved corresponding DNA damage repair mechanisms.However,there are always some lesions that cannot be repaired in time,which will remain on the genome until the S phase of the cell cycle,causing structural changes in the DNA double helix.These unusual DNA structures often stall the ongoing replication forks,thereby blocking genome replication.Restarting the replication fork requires the recruitment of specialized TLS polymerases to bypass the lesion before handing it back to the replicative polymerase to continue the genome replication.Cellular organisms often encode a variety of different types of DNA polymerases to ensure the faithful replication of the genome.For example,Sulfolobus(a model organism for studying DNA damage repair in thermophilic acidophilic Crenarchaea)encodes four DNA polymerases,including Dpol,Dpo2,Dpo3 of the B-family and Dpo4 of the Y-family.Among them,Dpo1 and Dpo3 may be mainly responsible for the routine replication of genome.Dpo4 can bypass multiple types of damage with different efficiencies in vitro,thus it had been employed as a model to study the mechanism of damage tolerance of Y-family DNA polymerases.However,in Sulfolobus,Dpo4 expression is constitutive,thus it may not play a major role in DNA damage repair upon the DNA damage response.Notably,the only damage-induced polymerase in Sulfolobus is Dpo2.Previous in vitro studies and bioinformatics analysis suggested that Dpo2 might not be active in DNA synthesis.Nevertheless,our genetic analysis demonstrated that it does play an important role in DNA damage repair.This thesis studied the conserved B-family DNA polymerase SisDpo2 in Sulfolobus,finding that this unusual enzyme is involved in translesion DNA synthesis(TLS)by using novel mechanisms.In order to study the function and mechanism of this protein in DNA damage tolerance,SisDpo2 was expressed in Sulfolobus islandicus REY15A,the native host,and purified into apparent homogeneity.First,primer extension experiments were conducted using undamaged DNA substrate,and it was revealed that the protein has a robust nucleotide incorporation activity,but exhibits poor processivity.The proofreading activity of the protein was subsequently tested using end-mismatched substrates,and it was found that the protein had no 3’-5’ exonuclease activity.The fidelity of SisDpo2 was further analyzed using steady-state kinetics,and it was indicated that it performs high fidelity nucleotide incorporation on undamaged DNA and extends the mismatched primer ends with high efficiency.Finally,primer extension experiments were conducted using various damaged substrates to analyze the translesion activity of the protein.It was revealed that SisDpo2 can efficiently extend the primer downstream the damage,compared with inefficient nucleotide incorporation activity at damage sites.Taken together,these studies indicated that SisDpo2 primarily functions as a mismatch and lesion extender,in analogy to the eukaryotic Pol ζ.Currently,Dpo2 is the only extender B-family DNA polymerase identified in prokaryotes and represents the only archaeal B-family DNA polymerase that is involved in TLS.Considering that Dpo4 can effectively perform nucleotide incorporation at the AP site which is one of the most abundant DNA lesions in cells,and downstream of which can be efficiently extended by Dpo2,this study examined whether they could act in concert during the bypass of AP site.It was revealed that neither SisDpo2 nor SisDpo4 can effectively bypass the AP site alone,however,their sequential action can promote the process.Then,steady-state kinetics was used to analyze the activity changes of both during the AP bypass.It was demonstrated that the activity of SisDpo4 is inhibited at positions 1-4 after the AP site,while SisDpo2 can effectively perform TLS extension.The activities of SisDpo2 and SisDpo4 were inhibited when the TLS patch(from the AP site)reached 5 nt and 6 nt,respectively,suggesting the occurrence of a switch from TLS polymerase to replicative DNA polymerase here.We further analyzed the activity changes of replicative polymerase SisDpol on the same substrates.Coincidentally,this happened to be the switching site for the polymerase activity and exonuclease activity of the SisDpo1.In summary,this study found that SisDpo2 may play a "bridging" role in TLS,which is responsible for filling the activity gap between SisDpo4 that functions in TLS insertion and SisDpo1 that conducts the high-fidelity genome replication after TLS.A novel TLS mechanism mediated by the B-family DNA polymerase Dpo2 in archaea was identified.