Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase eta.
DNA polymerase eta (Poleta) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a cis-syn thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Poleta. Poleta displayed "burst" kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (Kd) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (kpol) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.
Washington
M Todd
MT
Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061, USA.
Prakash
Louise
L
Prakash
Satya
S
D001483:Base Sequence; D001665:Binding Sites; D004260:DNA Repair; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D011740:Pyrimidine Dimers / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D000099270:Y-Family DNA Polymerases
D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase; D003854:Deoxyribonucleotides; D011740:Pyrimidine Dimers; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases