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 en vironment 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 spe ctroscopic probe of the mechanism of nucleotide incorporation by Esche richia coli Pol I Klenow fragment (KF) and bacteriophage T4 DNA polyme rase. The nucleotidyl transfer reaction may be monitored in real time by following the fluorescence of 2-AP, allowing the detection of trans ient intermediates along the reaction pathway that are inaccessible th rough traditional radioactive assays. Previous studies with Klenow fra gment [Kuchta, R. D., Mizrahi, V., Benkovic, P. A., Johnson, K. A., an d 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 nucle otide 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 m isincorporation reaction, and we present direct spectroscopic evidence for an activated KF'-DNA-dNTP species following the conformational ch ange step which features hydrogen bonding between the incoming and tem plate bases. In addition, we have utilized these same experiments to d emonstrate the existence of a similar nonchemical step in the mechanis m of dNTP incorporation by bacteriophage T4 DNA polymerase. This study provides the first direct evidence of a conformational change for T4 polymerase and emphasizes the importance of this step in a general pol ymerase kinetic sequence.