Cloning Expression And Biochemical Characterization Of Uracil-DNA Glycosylase IV And V From S. Acidocaldarius

Sulfolobus acidocaldarius is an aerobic thermoacidophilic crenarchaeon which grows optimally at 70~85°C and p H 2~4 in terrestrial solfataric springs, which often results in high frequency of base deamination. Cytosine d C and d A change into uracil and hypoxanthine d I. The damage base described above is the primary cause of base mutation. In the process of DNA replication, d U paired with d A and hypoxanthine can be paired with four kinds of bases, which resulting in mutations G: C â†' A: T and A: T â†' G: C/C: G/T: A,respectively. In order to adapt to such a high rate of deamination damage caused by high temperature environment, a perfect DNA repair systems of extreme temperature microbes have evolved to deal with these deamination damage. 1) d UTP hydrolysis by d UTPase, generate d UDP and phosphoric acid, eliminate harmful d UTP; 2) Uracil DNA glycosylase(uracil-DNA glycosylase, UDG) excised d U bases from damage DNA chain, and generate AP sites. After the removal of d U,other proteins participate in the base excision repair system complete the whole repair process. AP endonuclease cut off DNA on AP site,forming a gap of 3 ’hydroxyl and a 5’ phosphate; DNA polymerase added one or more correctly nucleotide in the gap; DNA ligase finished the entire base excision repair reaction. In order to clarify the base excision repair mechanism in extremely acidophilic thermophilic microorganisms. We successfully cloned the genes of udg IV and udg Vin S. Acidocaldarius. Identified its enzymatic properties of DNA glycosidase in vitro. The results confirms our speculation, two kinds of protein purified from S.aci shows the uracil DNA glycosylase activity.The optimal temperature and p H value for uracil excision by S.aci UDGs are 65 ℃ and 8.5,respectively. The removal of uracil is inhibited by the divalent ions of Ni2+, Cu2+, Co2+, Zn2+, as well as a high concentration of Na Cl over 0.05mol/L. The phosphorothioates near uracil strongly inhibit the excision of uracil by S.aci UDGs, indicating that the phosphate group near the damage d U bases does participated in the interactions between UDG and substrate DNA. S.aci UDGs excises uracil from various DNA substrates with the following order: U / C≥U/ T> U / G> ss DNA≥U / A. The specific activity of S.aci UDGIV and S.aci UDGV were 1*106 U/mg and 1333 U/mg, respectively. DNA polymerase have different effects on the catalytic activity of S.aci UDGIV and S.aci UDGV. Suggesting that binding mechanisms with substrate DNA may have differences between DNA polymerase and DNA glycosidase,or the activity and stability of DNA glycosidase may needs more assistance of repair proteins.