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

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, Ser⁸⁸³ 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 a is involved in primer interaction during DNA synthesis and plays an essential role in mispair extension fidelity of DNA synthesis.