Effect of accessory proteins on T4 DNA polymerase replication fidelity.
The influence of replication accessory proteins on the fidelity of T4 DNA polymerase has been examined. Steady-state kinetic measurements showed that exonuclease-deficient T4 DNA polymerase, alone or with clamp loaders gp44/gp62 and polymerase clamp gp45, displays decreased binding affinity for incorrect as compared to correct dNTPs and a deceased kcat for misinsertion as compared to correct insertion. Kinetic constants were similar with and without accessory proteins, indicating that accessory proteins had little effect on misinsertion. They also had little effect on the Km value for extension of a T.T mismatch. However, the kcat value for T.T mismatch extension was fivefold higher in the presence of the clamp loader and clamp proteins. Thus, in the absence of proofreading, these accessory proteins may promote stable misincorporation. The kinetic analysis is supported by error rate determinations during gap-filling synthesis, which require both misinsertion and mispair extension. For some mispairs, the accuracy of exonuclease-deficient polymerase alone is similar to that in the presence of clamp loader, clamp and single-stranded DNA binding protein (gp32). However, exonuclease-deficient holoenzyme complex is actually less accurate than the polymerase alone for some base substitutions. We suggest that gp45 promotes extension of mismatches by tethering the polymerase to DNA, a process that may be relevant to replication past lesions or other blocks to DNA synthesis. The error rate for one-nucleotide deletions in homopolymeric runs was similar for the polymerase with or without its accessory proteins. This implies that strand misalignment errors arise during highly processive replication. Thus, either unpaired bases can migrate through the run while the DNA polymerase is bound to the template-primer, or the DNA polymerase dissociates from the DNA to allow misalignment but remains tethered to the template through interactions with the clamp. Finally, the T4 replication accessory proteins reduced by >/=10-fold the rate at which exonuclease-deficient T4 DNA polymerase generated deletions of larger numbers of nucleotides, indicating that these proteins influence replication fidelity for other than single base mutations.
Kroutil
L C
LC
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Frey
M W
MW
Kaboord
B F
BF
Kunkel
T A
TA
Benkovic
S J
SJ
D017122:Bacteriophage T4 / Q000378:metabolism*; D001483:Base Sequence; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D016368:Frameshift Mutation; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D015534:Trans-Activators / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D015534:Trans-Activators; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C074855:gene 44 protein, Enterobacteria phage T4; C074555:gene 45 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4; D004259:DNA-Directed DNA Polymerase