Reversal of a mutator activity by a nearby fidelity-neutral substitution in the RB69 DNA polymerase binding pocket.
Phage RB69 B-family DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. Ser565 is a nearby conserved residue that also contributes to the binding pocket, but a S565G replacement has only a small impact on DNA replication fidelity. When Y567A and S565G replacements were combined, mutator activity was strongly decreased compared to that with Y567A replacement alone. Analyses conducted both in vivo and in vitro revealed that, compared to Y567A replacement alone, the double mutant mainly reduced base substitution mutations and, to a lesser extent, frameshift mutations. The decrease in mutation rates was not due to increased exonuclease activity. Based on measurements of DNA binding affinity, mismatch insertion, and mismatch extension, we propose that the recovered fidelity of the double mutant may result, in part, from an increased dissociation of the enzyme from DNA, followed by the binding of the same or another polymerase molecule in either exonuclease mode or polymerase mode. An additional antimutagenic factor may be a structural alteration in the polymerase binding pocket described in this article.
Trzemecka
Anna
A
Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-104 Warsaw, Poland.
Jacewicz
Agata
A
Carver
Geraldine T
GT
Drake
John W
JW
Bebenek
Anna
A
D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016368:Frameshift Mutation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020125:Mutation, Missense; D017900:Myoviridae / Q000201:enzymology*; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004279:DNA, Viral; D050505:Mutant Proteins; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase