The mechanism of nucleotide incorporation by human DNA polymerase eta differs from that of the yeast enzyme.
DNA polymerase eta (Poleta) catalyzes the efficient and accurate synthesis of DNA opposite cyclobutane pyrimidine dimers, and inactivation of Poleta in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Pre-steady-state kinetic studies of yeast Poleta have indicated that the low level of fidelity of this enzyme results from a poorly discriminating induced-fit mechanism. Here we examine the mechanistic basis of the low level of fidelity of human Poleta. Because the human and yeast enzymes behave similarly under steady-state conditions, we expected these enzymes to utilize similar mechanisms of nucleotide incorporation. Surprisingly, however, we find that human Poleta differs from the yeast enzyme in several important respects. The human enzyme has a 50-fold-faster rate of nucleotide incorporation than the yeast enzyme but binds the nucleotide with an approximately 50-fold-lower level of affinity. This lower level of binding affinity might provide a means of regulation whereby the human enzyme remains relatively inactive except when the cellular deoxynucleoside triphosphate concentrations are high, as may occur during DNA damage, thereby avoiding the mutagenic consequences arising from the inadvertent action of this enzyme during normal DNA replication.
Washington
M Todd
MT
Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061, USA.
Johnson
Robert E
RE
Prakash
Louise
L
Prakash
Satya
S
D001483:Base Sequence; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017384:Sequence Deletion; D013045:Species Specificity; D013379:Substrate Specificity; D014983:Xeroderma Pigmentosum / Q000201:enzymology / Q000235:genetics; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D009838:Oligodeoxyribonucleotides; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases