%0 Journal Article %J Biochemistry %D 2008 %T Biochemical evolution of DNA polymerase eta: properties of plant, human, and yeast proteins. %A Peter D Hoffman %A Curtis, Marc J %A Iwai, Shigenori %A John B Hays %K Amino Acid Sequence %K Arabidopsis %K Base Sequence %K Biochemical Phenomena %K Biochemistry %K Conserved Sequence %K DNA-Directed DNA Polymerase %K Evolution, Molecular %K Humans %K Kinetics %K Molecular Sequence Data %K Nucleotides %K Photochemistry %K Saccharomyces cerevisiae %K Sequence Alignment %X

To assess how evolution might have biochemically shaped DNA polymerase eta (Poleta) in plants, we expressed in Escherichia coli proteins from Arabidopsis thaliana (At), humans (Hs), and the yeast Saccharomyces cerevisiae (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 (80-110 mM salt). Specific activities for "standing-start" extension of synthetic primers ending opposite the second template nucleotide 3' to T-T were roughly similar. During subsequent "running-start" insertions past T-T and the next 5' ( N + 1) nucleotide, AtPoleta and HsPoleta appeared more processive, but DNA sequence contexts strongly affected termination probabilities. Lesion-bypass studies employed four different templates containing T[CPD]Ts, and two containing pyrimidine (6-4')-pyrimidinone photoproducts ([6-4]s). AtPoleta made the three successive insertions [opposite the T[CPD]T and (N + 1) nucleotides] that define bypass nearly as well as HsPoleta and somewhat better than ScPoleta. Again, sequence context effects were profound. Interestingly, the level of insertion opposite the ( N - 1) nucleotide 3' to T[CPD]T by HsPoleta and especially AtPoleta, but not ScPoleta, was reduced (up to 4-fold) relative to the level of insertion opposite the ( N - 1) nucleotide 3' to T-T. Evolutionary conservation of efficient T[CPD]T bypass by HsPoleta and AtPoleta may reflect a high degree of exposure of human skin and plants to solar UV-B radiation. The depressed ( N - 1) insertion upstream of T[CPD]T (but not T-T) may reduce the extent of gratuitous error-prone insertion.

%B Biochemistry %V 47 %P 4583-96 %8 2008 Apr 22 %G eng %N 16 %R 10.1021/bi701781p %0 Journal Article %J DNA Repair (Amst) %D 2005 %T Binding of MutS mismatch repair protein to DNA containing UV photoproducts, "mismatched" opposite Watson--Crick and novel nucleotides, in different DNA sequence contexts. %A Peter D Hoffman %A Wang, Huixian %A Christopher W Lawrence %A Iwai, Shigenori %A Hanaoka, Fumio %A John B Hays %K Adenosine Triphosphatases %K Amino Acid Sequence %K Bacterial Proteins %K Base Pair Mismatch %K Base Sequence %K DNA %K DNA Repair %K DNA-Binding Proteins %K Electrophoretic Mobility Shift Assay %K Molecular Sequence Data %K Mutagenesis %K MutS DNA Mismatch-Binding Protein %K Nucleotides %K Protein Binding %K Ultraviolet Rays %X

Mismatch-repair (MMR) systems suppress mutation via correction of DNA replication errors (base-mispairs) and responses to mutagenic DNA lesions. Selective binding of mismatched or damaged DNA by MutS-homolog proteins-bacterial MutS, eukaryotic MSH2.MSH6 (MutSalpha) and MSH2.MSH3-initiates mismatch-correction pathways and responses to lesions, and may cumulatively increase discrimination at downstream steps. MutS-homolog binding selectivity and the well-known but poorly understood effects of DNA-sequence contexts on recognition may thus be primary determinants of MMR specificity and efficiency. MMR processes that modulate UV mutagenesis might begin with selective binding by MutS homologs of "mismatched" T[CPD]T/AG and T[6--4]T/AG photoproducts, reported previously for hMutSalpha and described here for E. coli MutS protein. If MMR suppresses UV mutagenesis by acting directly on pre-mutagenic products of replicative bypass, mismatched photoproducts should be recognized in most DNA-sequence contexts. In three of four contexts tested here (three substantially different), T[CPD]T/AG was bound only slightly better by MutS than was T[CPD]T/AA or homoduplex DNA; only one of two contexts tested promoted selective binding of T[6--4]T/AG. Although the T:G pairs in T[CPD]T/AG and T/G both adopt wobble conformations, MutS bound T/G well in all contexts (K(1/2) 2.1--2.9 nM). Thus, MutS appears to select the two mismatches by different mechanisms. NMR analyses elsewhere suggest that in the (highly distorted) T[6--4]T/AG a forked H-bond between O2 of the 3' thymine and the ring 1-imino and exocyclic 2-amino guanine protons stabilizes a novel planar structure not possible in T[6--4]T/AA. Replacement of G by purines lacking one (inosine, 2-aminopurine) or both (nebularine) protons markedly reduced or eliminated selective MutS binding, as predicted. Previous studies and the work here, taken together, suggest that in only about half of DNA sequence contexts could MutS (and presumably MutSalpha) selectively bind mismatched UV photoproducts and directly suppress UV mutagenesis.

%B DNA Repair (Amst) %V 4 %P 983-93 %8 2005 Aug 15 %G eng %N 9 %R 10.1016/j.dnarep.2005.04.018