The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase.
The fluorescent properties and their sensitivity to the surrounding environment of the nucleotide analog 2-aminopurine (2-AP) have been well documented. In this paper we describe the use of 2-AP as a direct spectroscopic probe of the mechanism of nucleotide incorporation by Escherichia coli Pol I Klenow fragment (KF) and bacteriophage T4 DNA polymerase. The nucleotidyl transfer reaction may be monitored in real time by following the fluorescence of 2-AP, allowing the detection of transient intermediates along the reaction pathway that are inaccessible through traditional radioactive assays. Previous studies with Klenow fragment [Kuchta, R. D., Mizrahi, V., Benkovic, P. A., Johnson, K. A., & Benkovic, S. J. (1987) Biochemistry 26, 8410-8417] have revealed the presence of a nonchemical step prior to chemistry and have identified this conformational change as the rate-limiting step of correct nucleotide incorporation. During correct incorporation, phosphodiester bond formation occurs at a rate greater than the conformational change and has not been measured. However, during misinsertion, the rate of the chemical step becomes partially rate limiting and it becomes possible to detect both steps. We have successfully decoupled the chemical and conformational change steps for nucleotide insertion by KF using the misincorporation reaction, and we present direct spectroscopic evidence for an activated KF'-DNA-dNTP species following the conformational change step which features hydrogen bonding between the incoming and template bases. In addition, we have utilized these same experiments to demonstrate the existence of a similar nonchemical step in the mechanism of dNTP incorporation by bacteriophage T4 DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)
Frey
M W
MW
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Sowers
L C
LC
Millar
D P
DP
Benkovic
S J
SJ
D015075:2-Aminopurine / Q000737:chemistry*; D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D007700:Kinetics; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D013942:Thymine Nucleotides; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D015075:2-Aminopurine; D004247:DNA; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate