Roles of the polymerase and BRCT domains of Rev1 protein in translesion DNA synthesis in yeast in vivo.
Rev1p in yeast is essential for the translesion of abasic sites and 6-4 photoproducts. It plays a role as a translesion polymerase, but also supports translesion catalyzed by other polymerases. The protein has two domains, BRCT and Y-family polymerase. A point mutation in the BRCT domain is known to abolish the second function. In the present research, we have studied the effects of deletion of the BRCT domain and a point mutation at the two amino acids in the putative polymerase active center. We have introduced an abasic site, its tetrahydrofuran analog, and a 6-4 thymine-thymine photoproduct using the oligonucleotide transformation assay. Translesion efficiencies were estimated from the transforming activities of the oligonucleotides with a lesion, and the mutation spectra were analyzed by DNA sequencing of the transformants. Results showed that the lack of the BRCT domain reduced translesion efficiencies, but that substantial translesion synthesis took place. The mutation spectra of the lesions were not greatly affected. Therefore, the BRCT domain may be important, but dispensable for translesion synthesis. In contrast, the polymerase mutation, rev1AA, has only small effects on the translesion efficiencies, but the mutation spectra were greatly affected; the incorporation of dCMP opposite the lesions was specifically lost. This clearly shows that the polymerase domain is responsible for the dCMP incorporation. The effect of Poleta was also analyzed. From all the results DNA polymerases other than these two translesion polymerases, too, seem to initiate the translesion synthesis.
Otsuka
Chie
C
Department of Genomics and Proteomics, Okayama University Advanced Science Research Center, Tsushima, Okayama 700-8530, Japan.
Kunitomi
Nozomu
N
Iwai
Shigenori
S
Loakes
David
D
Negishi
Kazuo
K
D000595:Amino Acid Sequence; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage; D004252:DNA Mutational Analysis; D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008954:Models, Biological; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D000099270:Y-Family DNA Polymerases
D004247:DNA; D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase; D009713:Nucleotidyltransferases; D029701:Saccharomyces cerevisiae Proteins; C100951:REV1 protein, S cerevisiae; D000099270:Y-Family DNA Polymerases