Involvement of phenylalanine 272 of DNA polymerase beta in discriminating between correct and incorrect deoxynucleoside triphosphates.
DNA polymerase beta is a small monomeric polymerase that participates in base excision repair and meiosis [Sobol, R., et al. (1996) Nature 379, 183-186; Plug, A., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1327-1331]. A DNA polymerase beta mutator mutant, F272L, was identified by an in vivo genetic screen [Washington, S., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1321-1326]. Residue 272 is located within the deoxynucleoside triphosphate (dNTP) binding pocket of DNA polymerase beta according to the known DNA polymerase beta crystal structures [Pelletier, H., et al. (1994) Science 264, 1891-1893; Sawaya, M., et al. (1997) Biochemistry 36, 11205-11215]. The F272L mutant produces errors at a frequency 10-fold higher than that of wild type in vivo and in the in vitro HSV-tk gap-filling assay. F272L shows an increase in the frequency of both base substitution mutations and frameshift mutations. Single-enzyme turnover studies of misincorporation by wild type and F272L DNA polymerase beta demonstrate that there is a 4-fold decrease in fidelity of the mutant as compared to that of the wild type enzyme for a G:A mismatch. The decreased fidelity is due primarily to decreased discrimination between the correct and incorrect dNTP during ground-state binding. These results suggest that the phenylalanine 272 residue is critical for maintaining fidelity during the binding of the dNTP.
Li
S X
SX
Departments of Therapeutic Radiology and Genetics, and Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Vaccaro
J A
JA
Sweasy
J B
JB
D020137:Base Pair Mismatch; D001483:Base Sequence; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D011994:Recombinant Proteins; D010649:Phenylalanine; D019951:DNA Polymerase beta