Activity of Human DNA Polymerases α and β 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 α and β (Hpolα, Hpolβ) 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 Hpolα, and 26-153% with Hpolβ. The concentrations of CldATP for half maximum velocity is 0.6 μM for Hpolα, and 6 μM for Hpolβ, each about twice the value for dATP. Thus, CldATP is a good substrate for both enzymes but is more efficiently used by Hpolα. Addition of a single analogue residue by Hpolβ 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 Hpolα 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.