A single stranded DNA binding protein isolated from HeLa cells facilitates Ni2+ activation of DNA polymerases in vitro.
The divalent nickel ion (Ni2+) is one of several metal ions that can substitute for Mg2+ in the activation of DNA polymerases in vitro, but usually with very low efficiency. We have purified and partially characterized a Ni(2+)-binding protein (p40) from HeLa cell extracts that can specifically enhance the polymerase activity of DNA polymerase alpha (pol alpha) and other DNA polymerases in response to Ni2+. This protein, with a molecular mass of 40 kDa, is a single stranded DNA binding protein that binds to a M13 DNA template-primer with an optimum stoichiometry of approximately 90 equiv of protein per equiv of DNA template and enhances the affinity of pol alpha for the primer-template. In the presence of Ni2+, p40 exhibits an increased affinity for DNA. The p40 increased by 3- to 6-fold the rates at which pol alpha and the Klenow fragment of Escherichia coli DNA polymerase I (KF) replicate different DNA templates in response to Ni2+. The low processivity of Ni(2+)-activated pol on primed M13 ssDNA was also enhanced by the presence of p40. The rates of Ni(2+)-dependent replication by inherently more processive enzymes, DNA polymerase delta and T4 DNA polymerase, were not significantly increased by p40 when M13 ssDNA was used as a template; however, p40 did increase the activity of T4 polymerase on an activated calf thymus DNA template. The protein did not stimulate Mg(2+)-activated DNA replication.
Chin
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Department of Environmental Medicine, New York University Medical Center, Tuxedo 10987.
Snow
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Christie
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D004257:DNA Polymerase II / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells / Q000737:chemistry*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009532:Nickel / Q000378:metabolism / Q000494:pharmacology*; D013698:Templates, Genetic; D013938:Thymidine Monophosphate / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D013938:Thymidine Monophosphate; D009532:Nickel; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium