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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. 1993

S K Chunduru, and J R Appleman, and R L Blakley
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009711 Nucleotides The monomeric units from which DNA or RNA polymers are constructed. They consist of a purine or pyrimidine base, a pentose sugar, and a phosphate group. (From King & Stansfield, A Dictionary of Genetics, 4th ed) Nucleotide
D003838 Deoxyadenine Nucleotides Adenine nucleotides which contain deoxyribose as the sugar moiety. Deoxyadenosine Phosphates,Nucleotides, Deoxyadenine,Phosphates, Deoxyadenosine
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004256 DNA Polymerase I A DNA-dependent DNA polymerase characterized in prokaryotes and may be present in higher organisms. It has both 3'-5' and 5'-3' exonuclease activity, but cannot use native double-stranded DNA as template-primer. It is not inhibited by sulfhydryl reagents and is active in both DNA synthesis and repair. DNA Polymerase alpha,DNA-Dependent DNA Polymerase I,Klenow Fragment,DNA Pol I,DNA Dependent DNA Polymerase I,Polymerase alpha, DNA
D004257 DNA Polymerase II A DNA-dependent DNA polymerase characterized in E. coli and other lower organisms. It may be present in higher organisms and has an intrinsic molecular activity only 5% of that of DNA Polymerase I. This polymerase has 3'-5' exonuclease activity, is effective only on duplex DNA with gaps or single-strand ends of less than 100 nucleotides as template, and is inhibited by sulfhydryl reagents. DNA Polymerase epsilon,DNA-Dependent DNA Polymerase II,DNA Pol II,DNA Dependent DNA Polymerase II
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D013379 Substrate Specificity A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts. Specificities, Substrate,Specificity, Substrate,Substrate Specificities

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