Biochemical Characterization Of Translesl On Synthesis By Sulfolobus Acidocaldarius DNA Polymerases

Genetic information carrier DNA molecules are the basis of heredity and variation in living life.DNA stability mainly depends on the efficiency and completeness of DNA replication and DNA damage repair system.DNA polymerases are the enzymatic workhorses that ensure effective genome duplication and translesion synthesis,and crucial to cell survival.All the DNA polymerases possess DNA-dependent DNA polymerase activity,catalyzing DNA synthesis.Part of them own 3'to 5'exonuclease activities,can remove the mismatch nucleotides mis-incorporated by polymerases,performing the function of error correction.DNA molecules are labile at high temperatures,so hyperthermophiles face a bigger burden to maintain the genome integrity in which DNA polymerases play the most important role.To study the DNA synthesis mechanism of Sulfolobus acidocaldarius,a thermophilic species from Crenarchaeota,two B family DNA polymerases of polB1 and polB3,and one Y family DNA polymerase of polIV were recombinantly expressed,purified and biochemically characterized.Both DNA polymerases polB1and polB3 possessed DNA polymerase and 3'to 5'exonuclease activities;however,both activities of B3 were very inefficient in vitro.The polIV?Dbh?was a polymerase,not exonuclease.The activities of all the three DNA polymerases were dependent on divalent metal ions Mn²⁺and Mg²⁺.the highest activity at pH values ranging from 8.0 to 9.5.Their activities were inhibited by high concentration KCl.The optimal reaction temperatures for the three DNA polymerases were between 60 and 70°C.Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family.While the Y family DNA polymerase bypassed the two AP site analogues dSpacer and propane on template efficiently.These results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.After certified the translesion ability of Y family DNA polymerase polIV,we systematically explored the efficiency of Dbh bypassing various artificial spacers,the analogue of abasic sites,including different lengths of chain alkanes,polyethylene glycolchain,DNA,etc.Our results show that the molecular structure of Spacer?including the Spacer length,position,the difference of molecular configuration,etc.?,determines the TLS efficiency of polIV.With the increase of the length and number of Spacers,the TLS efficiency continuously reduced.DNA polymerase IV mainly adopts template sliding model performing TLS.In the process of TLS,incorporation of the first downstream nucleotide after damage sites is the rate-limiting step.According to the related crystal structure and molecular docking simulation analysis results,we identified the key amino acids residues specifically responsible for bypassing the spacers consisted of hydrocarbon chain by gene cloning and site-directed mutation.It's speculated that the interactions between carbons and three hydrophobic residues of Y249,R333,and I295 contribute to bypassing the spacers of hydrocarbon chains,furtherly confirmed the little finger domain of Y family polymerase plays the key role in TLS process.