Role of lysine 758 of Escherichia coli DNA polymerase I as assessed by site-directed mutagenesis.
Lys-758 of Escherichia coli DNA polymerase I has been implicated in the process of substrate dNTP binding (Basu, A., and Modak, M. J. (1987) Biochemistry 26, 1704-1709). To confirm and define the role of Lys-758 in the catalytic mechanism, we carried out site-directed mutagenesis of this residue. Catalytic activity of the purified mutant enzymes, K758A and K758R, showed severe reduction in the polymerase activity but little difference in the 3'-->5' exonuclease activity. Most interestingly, the catalytic ability of both mutant enzymes was maximally affected (300-1,000-fold decrease in kcat) with poly(dA).(dT)15 as template-primer (TP), whereas the ability to use poly(dC) templates decreased by only 20-fold in K758A and remained nearly unchanged with K758R. Kinetic characterization showed that Km(dNTP) increased moderately only with K758A, whereas Kd(TP) remained unchanged for both the mutants. However, binary complex formation between K758A and dNTP, but not between K758A and TP, was severely reduced. Analysis of the processive mode of DNA synthesis by K758A indicated that the mutant enzyme pauses at dA bases but does not dissociate from TP, suggesting a defect in its translocation ability. Thus, Lys-758 in polymerase I appears to participate in two distinct functions: (a) it facilitates the dNTP binding, and (b) it is required for the translocation along the template polynucleotide.
Pandey
V N
VN
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry-New Jersey Medical School, Newark 07103.
Kaushik
N
N
Modak
M J
MJ
D001483:Base Sequence; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D013698:Templates, Genetic
D017931:DNA Primers; D004256:DNA Polymerase I; D008239:Lysine