The long term goal of this project is to understand the molecular mechanism of DNA damage-induced mutagenesis in the eukaryotic cell. As a model system we will study the yeast, Saccharomyces cerevisiae. Genetic evidence indicates that the yeast Rev1, Rev3 and Rev7 proteins are required for DNA damage-induced mutagenesis. We have recently purified each of these proteins. The Rev3 and Rev7 proteins form a complex that has DNA polymerase activity. This enzyme, DNA polymerase zeta, has a superior ability to replicate past a T-T dimer in the template. The effect of the yeast replication factors RF-A, RF-C and PCNA on the activity of DNA polymerase zeta will be examined, with both normal and lesion-containing templates. The extension efficiency will be measured for primers containing different 3' mismatches, opposite both normal nucleotides and a TT dimer or an abasic site in the template. The Km for the interaction of polymerase zeta with these mismatched primer-templates will also be determined and compared with values for interaction with paired primers. The Rev1 protein is a deoxycytidyl terminal transferase that inserts a dCMP residue opposite an abasic site in the template. It is not clear how this activity could function in the bypass of UV-induced lesions. To investigate the possibility that the Rev1 protein is bifunctional, with one domain that functions in the bypass of UV lesions, perhaps by binding to the damage, and a terminal transferase domain that functions in bypass of abasic sites, we will construct various deletion and point mutants and test them for transferase activity and for complementation of the """"""""non-UV-mutable"""""""" rev1 mutant phenotype.We will also examine the ability of the Rev1 protein to band to normal and UV irradiated DNA and to enhance the bypass of UV lesions by Polymerase zeta. Finally, using several methods, including a yeast two-hybrid screen, we will search for other subunits or interacting proteins in order to define the native structure of these two enzymes.
| Mouro, I; Halleck, M S; Schlegel, R A et al. (1999) Cloning, expression, and chromosomal mapping of a human ATPase II gene, member of the third subfamily of P-type ATPases and orthologous to the presumed bovine and murine aminophospholipid translocase. Biochem Biophys Res Commun 257:333-9 |
| Vaisman, A; Lim, S E; Patrick, S M et al. (1999) Effect of DNA polymerases and high mobility group protein 1 on the carrier ligand specificity for translesion synthesis past platinum-DNA adducts. Biochemistry 38:11026-39 |
