MUTATIONAL STUDIES OF HUMAN DNA POLYMERASE-ALPHA - SERINE 867 IN THE 2ND MOST CONSERVED REGION AMONG ALPHA-LIKE DNA-POLYMERASES IS INVOLVEDIN PRIMER BINDING AND MISPAIR PRIMER EXTENSION

The second most conserved region of alpha-like DNA polymerases, region II, spans a block of 40 amino acid residues centered at the core sequ ence -DFNSLYPSII-. In the previous paper, we described mutational stud ies of 3 amino acid residues in region II which includes 2 amino acid residues in the core sequence. We showed that residues Asp860 and Tyr8 65 in the core sequence are involved in substrate deoxynucleotide trip hosphate (dNTP) binding. We further showed that the phenyl moiety of t he Tyr865 side chain interacts with the incoming dNTP and is responsib le for the misinsertion fidelity of the enzyme. In this report, we inv estigated the function of 2 serine residues, Ser863 and Ser867, in thi s core sequence. Mutation of these 2 Ser residues to either Ala or Thr yielded mutant enzymes with similar K(m) for dNTPs, k(cat), processiv ity, and misinsertion fidelity of DNA synthesis as the wild type enzym e. 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 prime r-template. DNA footprinting experiments of primer with the dideoxynuc leotide terminus indicated that the structural feature of the primer r ecognized by Ser867 is the 3'-OH terminus. Single-stranded DNA inhibit ion data suggest that removal of the hydroxyl side chain of Ser867 aff ects the polymerase's interaction with primer and not with template. M utation of Ser867 to Ala also decreases the mutant enzyme's K(m) for d NTP to extend a mispaired primer and thus enhances its capacity to ext end a mispaired primer terminus. These data support the conclusion tha t the hydroxyl side chain of Ser867 of human DNA polymerase alpha is i nvolved in primer interaction during DNA synthesis and plays an essent ial role in mispair extension fidelity of DNA synthesis.