Processivity clamp gp45 and ssDNA-binding-protein gp32 modulate the fidelity of bacteriophage RB69 DNA polymerase in a sequence-specific manner, sometimes enhancing and sometimes compromising accuracy.
Numerous studies of the impact of accessory proteins upon the fidelity of DNA synthesis have provided a complex and sometimes discordant picture. We previously described such an analysis conducted in vitro using various bacteriophage RB69 gp43 mutator DNA polymerases with or without the accessory proteins gp32 (which binds single-stranded DNA) plus gp45/44/62 (processivity clamp and its loaders). Mutations were scored at many sites in the lacZalpha mutation reporter sequence. Unexpectedly, the accessory proteins sometimes decreased and sometimes increased fidelity at a handful of specific sites. Here, we enlarge our analysis with one particular mutator polymerase compromised in both insertion accuracy and proofreading and also extend the analysis to reactions supplemented only with gp32 or only with gp45/44/62. An overall 1.56-fold increase in mutation frequencies was produced by adding single or multiple accessory proteins and was driven mainly by increased T(template)*G(primer) mispairs. Evidence was found for many additional sites where the accessory proteins influence fidelity, indicating the generality of the effect. Thus, accessory proteins contribute to the site-specific variability in mutation rates characteristically seen in mutational spectra.
Bebenek
Anna
A
Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw.
Carver
Geraldine T
GT
Kadyrov
Farid A
FA
Kissling
Grace E
GE
Drake
John W
JW
D017122:Bacteriophage T4 / Q000737:chemistry*; D001435:Bacteriophages / Q000378:metabolism; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology*; D017930:Genes, Reporter; D007763:Lac Operon; D046911:Macromolecular Substances / Q000737:chemistry; D008957:Models, Genetic; D009010:Monte Carlo Method; D009154:Mutation; D011485:Protein Binding; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology*; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D015534:Trans-Activators; D014764:Viral Proteins; C074555:gene 45 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase