Mechanism Of Thermococcus Eurythermalis Endonuclease ? Cleaving AP Site And Various Analogues In DNA

The genome integrity depends on the fidelity of DNA replication and repair.DNA damage is the changes of nucleotides and DNA structure caused by physical and chemical factors in cell and environment,and it will bring some potential mutagenic and toxic effects on cell.Among all DNA damages,AP sites(apyrimidinic/apurinic sites)are the most common lesion.AP sites cannot guide the incorporation of a correct(d)NMP because of no bases,and then it will inhibit replication or transcription,or lead to base mutation.In cell,AP sites are mainly repaired by the base excision repair(BER)pathway.In BER,the enzymes important for cleaving DNA phosphodiester backbone at AP sites are called AP endonuclease.The AP endonucleases,cleave the 5'-side of AP site through hydrolysis of phosphodiester bond to yield a free 3'-OH and a 5'-deoxyribose-phosphate(d RP),including exonuclease III and endonuclease ?.Endonuclease ? is a DNA damage-specific endonuclease and mainly hydrolyzes the phosphodiester bond at the 5'-side of an AP site in DNA.Endonuclease ? also possesses 3'-exonuclease activity for removing DNA3'-blocking groups and normal nucleotide.The extremely thermophilic microorganisms are faced with more AP site damages.Therefore the role of AP endonucleases is even more important.In addition,endonuclease ? has a wider distribution range than exonuclease III in archaea.Since repairing AP site is very important for hyperthermophilic archaea,this study used T.eurythermalis endonuclease ?(Teuendo ?)as a research object to explore its enzymatic characteristics and cleavage mechanism.Our research is focused on the cleavage of Teuendo ? on AP sites and their analogues.Here,our results shows that Teuendo ? possesses the AP endonuclease and 3'-exonuclease activities,and prefers Mg²⁺,not Zn²⁺as seen in E.coli endonuclease ?,as cofactor.Besides natural AP site deoxyribose,it could hydrolyze the phosphodiester bond at the 5'-side of various AP site analogues,including alkane Spacer and polyethylene glycol Spacer,with a preference to the Spacers having a three-carbon main chain.In addition,the position of AP site strongly affects hydrolysis reaction.Teuendo ? could remove one or more consecutive AP sites at the3'-terminus.However,the Spacer at the 5'-terminus could not be removed,and their efficient removal requires that?two normal nucleotides exist between the 5'-terminus and the Spacer.To further investigate the cleavage mechanism of Teuendo ?,we constructed a series of Teuendo ? mutants and modeled its structure.The results indicate that the key residues for binding substrates and coordinating metal ion cofactor exert an important role in hydrolyzing the phosphodiester bond.Compared with the residue penetrating the minor groove,the residues for binding AP site containing strand and complementary strand are more important for the hydrolysis of phosphodiester bond.The structure modeling results show that the topological structure of Teuendo ? includes9?-helix,5 recognition loops(R-loop),and 8?-strands.The superposition of modeled Teuendo ? structure and the crystal structure of Ecoendo ? indicates that Teuendo ? has a more disordered N-terminal domain than Ecoendo ?.Our results provide the biochemical basis for repairing AP sites in hyperthermophile cell.