Yeast pol eta holds a cis-syn thymine dimer loosely in the active site during elongation opposite the 3'-T of the dimer, but tightly opposite the 5'-T.
Polymerase eta is a member of the Y family of DNA polymerases which is able to bypass thymine dimers efficiently and in a relatively error-free manner. To elucidate the mechanism of dimer bypass, the efficiency of dAMP and pyrene nucleotide insertion opposite the thymine dimer and its N3-methyl derivatives was determined. Pol eta inserts pyrene nucleotide with greater efficiency than dAMP opposite the 3'-T of an undimerized or dimerized T and is an effective inhibitor of DNA synthesis by pol eta. Substitution of the N3H of the 3'-T of an undimerized T or a dimerized T with a methyl group has little effect on the insertion efficiency of pyrene nucleotide but greatly inhibits the insertion of dAMP. Together, these results suggest that the error-free insertion of dAMP opposite the 3'-T of the cis-syn thymine dimer happens by way of a loosely held dimer in the active site which can be displaced from the active site by pyrene nucleotide. In contrast, pol eta cannot insert pyrene nucleotide opposite the 5'-T of the dimer, whereas it can insert dAMP with efficiency comparable to that opposite the 3'-T. The inability to insert pyrene nucleotide opposite the 5'-T of the dimer is consistent with the idea that while the polymerase binds loosely to a templating nucleotide, it binds tightly to the nucleotide to its 3'-side. Overall, the results show a marked difference from similar studies on pol I family polymerases, and suggest mechanisms by which this Y family polymerase can process damaged DNA efficiently.
Sun
Liping
L
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Zhang
Kaijiang
K
Zhou
Lilly
L
Hohler
Paul
P
Kool
Eric T
ET
Yuan
Fenghua
F
Wang
Zhigang
Z
Taylor
John Stephen
JS
D001080:Apurinic Acid / Q000378:metabolism; D001665:Binding Sites; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011119:Polynucleotides / Q000378:metabolism; D011740:Pyrimidine Dimers / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D000099270:Y-Family DNA Polymerases
D001080:Apurinic Acid; D017931:DNA Primers; D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase; D003854:Deoxyribonucleotides; D011119:Polynucleotides; D011740:Pyrimidine Dimers; C001790:apyrimidinic acid; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases