Activity of human DNA polymerases alpha and beta with 2-chloro-2'-deoxyadenosine 5'-triphosphate as a substrate and quantitative effects of incorporation on chain extension.
When 2-chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) is incorporated into DNA by human polymerases alpha and beta (Hpol alpha, Hpol beta) the rate of chain extension decreases. In the present study primer extension has been quantitated by estimating the concentration of each successive oligonucleotide product at a series of time points. This has permitted calculation of pseudo-first-order rate constants for successive nucleotide additions to primer. By this method it has been shown that rate constants for CldATP addition are 79-100% of those for dATP in the case of Hpol alpha, and 26-153% with Hpol beta. The concentrations of CldATP for half maximum velocity is 0.6 microM for Hpol alpha, and 6 microM for Hpol beta, each about twice the value for dATP. Thus, CldATP is a good substrate for both enzymes but is more efficiently used by Hpol alpha. Addition of a single analogue residue by Hpol beta to any of seven primers decreases the rate constant for addition of the next nucleotide to 2-7% of that after dAMP addition and further extension is negligible. Consecutive additions of analogue residues by Hpol alpha progressively decrease the rate of subsequent extension, and after five consecutive additions extension virtually terminates. These effects probably make a major contribution to the cytotoxicity of chlorodeoxyadenosine and its therapeutic usefulness as an antileukemic agent.
Chunduru
S K
SK
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Appleman
J R
JR
Blakley
R L
RL
D001483:Base Sequence; D004247:DNA / Q000378:metabolism* / Q000494:pharmacology; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D013379:Substrate Specificity
D003838:Deoxyadenine Nucleotides; D009711:Nucleotides; C057381:2-chloro-2'-deoxyadenosine triphosphate; D004247:DNA; D004256:DNA Polymerase I; D004257:DNA Polymerase II; C026600:2'-deoxyadenosine triphosphate