Human DNA polymerase kappa bypasses and extends beyond thymine glycols during translesion synthesis in vitro, preferentially incorporating correct nucleotides.
Human polymerase kappa (polkappa), the product of the human POLK (DINB1) gene, is a member of the Y superfamily of DNA polymerases that support replicative bypass of chemically modified DNA bases (Ohmori, H., Friedberg, E. C., Fuchs, R. P., Goodman, M. F., Hanaoka, F., Hinkle, D., Kunkel, T. A., Lawrence, C. W., Livneh, Z., Nohmi, T., Prakash, L., Prakash, S., Todo, T., Walker, G. C., Wang, Z., and Woodgate, R. (2001) Mol. Cell 8, 7-8; Gerlach, V. L., Aravind, L., Gotway, G., Schultz, R. A., Koonin, E. V., and Friedberg, E. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11922-11927). Polkappa is shown here to bypass 5,6-dihydro-5,6-dihydroxythymine (thymine glycol) generated in two different DNA substrate preparations. Polkappa inserts the correct base adenine opposite thymine glycol in preference to the other three bases. Additionally, the enzyme correctly extends beyond the site of the thymine glycol lesion when presented with adenine opposite thymine glycol at the primer terminus. However, steady state kinetic analysis of nucleotides incorporated opposite thymine glycol demonstrates different misincorporation rates for guanine with each of the two DNA substrates. The two substrates differ only in the relative proportions of thymine glycol stereoisomers, suggesting that polkappa distinguishes among stereoisomers and exhibits reduced discrimination between purines when incorporating a base opposite a 5R thymine glycol stereoisomer. When extending beyond the site of the lesion, the misincorporation rate of polkappa for each of the three incorrect nucleotides (adenine, guanine, and thymine) is dramatically increased. Our findings suggest a role for polkappa in both nonmutagenic and mutagenic bypass of oxidative damage.
Fischhaber
Paula L
PL
Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9072, USA.
Gerlach
Valerie L
VL
Feaver
William J
WJ
Hatahet
Zafer
Z
Wallace
Susan S
SS
Friedberg
Errol C
EC
D000225:Adenine / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D009690:Nucleic Acid Conformation; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic; D013941:Thymine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D000225:Adenine; D004247:DNA; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D011506:Proteins; D013941:Thymine; C029389:thymine glycol; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases