Biochemical Evolution of DNA Polymerase η: Properties of Plant, Human, and Yeast Proteins.

To assess how evolution might have biochemically shaped DNA polymerase 1/ (Polt/) in plants. we expressed in E.scherichia co/i proteins from .4rabidopsis thu/lana (At), humans (Hs). and the yeast Saecharomycea cereuisiae (Sc), purified them to near homogeneity, and compared their properties. Consistent with the multiple divergent amino acids within mostly conserved polymerase domains, the polymerases showed modest, appreciable, and marked differences. respectively, in salt and temperature optima for activity and thermostability. We compared abilities to extend synthetic primers past template cyclobutane thymine dimers (T[CPD]T) or undamaged T-T under physiological conditions (80-I It) mM salt). Specific activities for "standing-start" extension of synthetic primers ending opposite the second template nucleotide 3' to T-T were roughly similar. DUriTig subsequent "running-start" insertions past T-T and the next 5' (N + I) nucleotide, AtPolt7 and HsPolij appeared more processive. but DNA sequence contexts strongly affected termination probabilities. Lesion-bypass studies employed four different templates containing TICPD]Ts, and two containing pyrimidine (6--t')-pyrimidinone photoproducts (16-415). AtPol~ made the three successive insertions [opposite the TICPD]T and (N + I) nucleotidesi that define bypass nearly as well as HsPoltj and somewhat better than ScPol~. Again, sequence context elfects were profound. Interestingly, the level of insertion opposite the (N - I) nucleotide 3' to TICPD1T by HsPolt/ and especially AtPolsi, but not ScPolli. was reduced (up to 4-fold) relative to the level of insertion opposite the (N - I) nucleotide 3' to T-T. Evolutionary conservation of efficient T[CPD]T bypass by HsPolq and AtPols~ may reflect a high degree of exposure of human skin and plants to solar UV-B radiation. The depressed (N - I) insertion upstream of T[CPDIT (but not T-T) may reduce the extent of gratuitous error-prone insertion. [ABSTRACT FROM AUTHOR]