Mutational studies of human DNA polymerase α. Serine 867 in the second most conserved region among α-like DNA polymerases is involved in primer binding and mispair primer extension

Q. Dong, W. C. Copeland, T. S.F. Wang

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

The second most conserved region of α-like DNA polymerases, region II, spans a block of 40 amino acid residues centered at the core sequence - DFNSLYPSII-. In the previous paper, we described mutational studies of 3 amino acid residues in region II which includes 2 amino acid residues in the core sequence. We showed that residues Asp860 and Tyr865 in the core sequence are involved in substrate deoxynucleotide triphosphate (dNTP) binding. We further showed that the phenyl moiety of the Tyr865 side chain interacts with the incoming dNTP and is responsible for the misinsertion fidelity of the enzyme. In this report, we investigated the function of 2 serine residues, Ser863 and Ser867, in this core sequence. Mutation of these 2 Ser residues to either Ala or Thr yielded mutant enzymes with similar K(m) for dNTPs, k(cat), processivity, and misinsertion fidelity of DNA synthesis as the wild type enzyme. However, mutation of Ser867 to Ala demonstrated a 30-fold increase in K(m) for primer-template and a 5-fold higher K(D) for binding primer-template. DNA footprinting experiments of primer with the dideoxynucleotide terminus indicated that the structural feature of the primer recognized by Ser867 is the 3'-OH terminus. Single- stranded DNA inhibition data suggest that removal of the hydroxyl side chain of Ser867 affects the polymerase's interaction with primer and not with template. Mutation of Ser867 to Ala also decreases the mutant enzyme's K(m) for dNTP to extend a mispaired primer and thus enhances its capacity to extend a mispaired primer terminus. These data support the conclusion that the hydroxyl side chain of Ser867 of human DNA polymerase α is involved in primer interaction during DNA synthesis and plays an essential role in mispair extension fidelity of DNA synthesis.

Original languageEnglish (US)
Pages (from-to)24175-24182
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number32
StatePublished - 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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