Translesion synthesis by yeast DNA polymerase zeta from templates containing lesions of ultraviolet radiation and acetylaminofluorene.
In the yeast Saccharomyces cerevisiae, DNA polymerase zeta (Polzeta) is required in a major lesion bypass pathway. To help understand the role of Polzeta in lesion bypass, we have performed in vitro biochemical analyses of this polymerase in response to several DNA lesions. Purified yeast Polzeta performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3' T, and predominantly A opposite the 5' T. Purified yeast Polzeta predominantly incorporated a G opposite an acetylaminofluorene (AAF)-adducted guanine. The lesion, however, significantly inhibited subsequent extension. Furthermore, yeast Polzeta catalyzed extension DNA synthesis from primers annealed opposite the AAF-guanine and the 3' T of the TT (6-4) photoproduct with varying efficiencies. Extension synthesis was more efficient when A or C was opposite the AAF-guanine, and when G was opposite the 3' T of the TT (6-4) photoproduct. In contrast, the 3' T of a cis-syn TT dimer completely blocked purified yeast Polzeta, whereas the 5' T was readily bypassed. These results support the following dual-function model of Polzeta. First, Polzeta catalyzes nucleotide incorporation opposite AAF-guanine and TT (6-4) photoproduct with a limited efficiency. Secondly, more efficient bypass of these lesions may require nucleotide incorporation by other DNA polymerases followed by extension DNA synthesis by Polzeta.
Guo
D
D
Graduate Center for Toxicology, 306 Health Sciences Research Building, University of Kentucky, Lexington, KY 40536, USA.
Wu
X
X
Rajpal
D K
DK
Taylor
J S
JS
Wang
Z
Z
D015073:2-Acetylaminofluorene / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D018736:DNA Adducts / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage / Q000235:genetics / Q000528:radiation effects*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis / Q000235:genetics; D011740:Pyrimidine Dimers / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013698:Templates, Genetic; D014466:Ultraviolet Rays
D018736:DNA Adducts; D005656:Fungal Proteins; D011740:Pyrimidine Dimers; C092219:REV7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006147:Guanine; D004247:DNA; D015073:2-Acetylaminofluorene; C425929:DNA polymerase zeta; D004259:DNA-Directed DNA Polymerase; C092602:REV3 protein, S cerevisiae