Properties of tyrosine 766-->serine mutant of Escherichia coli DNA polymerase I: template-specific effects.
In order to determine the role of Tyr 766 of Escherichia coli DNA polymerase I in the catalysis of DNA synthesis, we investigated the properties of a Tyr 766-->Ser (Y766S) mutant of the Klenow fragment of E. coli DNA polymerase I. We found that the rates of incorporation of only dTTP but not the other dNTP substrates were affected in the reactions catalyzed by the mutant enzyme, when homopolymeric template-primers were used. The mutant enzyme exhibited a reduced rate of synthesis only with poly(rA)- or poly(dA)-directed reactions. Examination of the ability of the mutant and the wild-type enzymes to bind to dGTP and dTTP, as judged by UV-mediated cross-linking, indicated nearly identical binding efficiencies of both nucleotides. However, the ability of the mutant enzyme to bind to poly(rA).(dT)15 and poly(dA).(dT)15 was found to be significantly reduced as compared to the binding to heteropolymeric DNA. In order to further define the nature of template-mediated restriction on the catalytic activity of the mutant enzyme, its ability to copy DNA templates containing a stretch of AAAAA and ACACA sequences was compared. The results show that DNA synthesis catalyzed by the mutant enzyme is significantly retarded when it encounters the AAAAA region of the template but not the ACACA region. Product analysis of the reaction directed by the two template-primers showed that the mutant enzyme stalls/terminates synthesis upon encountering an AAAAA sequence in the template.(ABSTRACT TRUNCATED AT 250 WORDS)
Desai
S D
SD
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry-New Jersey Medical School, Newark 07103-2714.
Pandey
V N
VN
Modak
M J
MJ
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis*; D018736:DNA Adducts; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics; D014466:Ultraviolet Rays
D018736:DNA Adducts; D017931:DNA Primers; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D013942:Thymine Nucleotides; D014443:Tyrosine; D012694:Serine; C029603:deoxyguanosine triphosphate; D004247:DNA; D004256:DNA Polymerase I; C024157:thymidine 5'-triphosphate