Effect of accessory proteins on T4 DNA polymerase replication fidelity.

Abstract:

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.

Polymerases:

Topics:

Fidelity, Accessory Proteins/Complexes, Nucleotide Incorporation, Exonuclease Activity

One line summary:

Lack of gp45 will significantly reduce the rate of replication, addition of other accessory proteins reduces the affinity for misinsertions.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 D219A Kroutil LC1998 3-5' Exonuclease (proofreading) No
T4 D219A Kroutil LC1998 5-3' Exonuclease Unspecified
T4 D219A Kroutil LC1998 Overall Error Rate 1.2E-05 errors/bp
T4 D219A Kroutil LC1998 Nucleotide Substitution Rate 4E-05 errors/bp Technique: M13mp2 forward mutation assay
T4 D219A Kroutil LC1998 Nucleotide Substitution Rate 0.0001 errors/bp Technique: M13mp2 opal codon reversion assay
T4 Kroutil LC1998 3-5' Exonuclease (proofreading) Yes
T4 Kroutil LC1998 5-3' Exonuclease Unspecified
T4 Kroutil LC1998 Frameshift Error Rate 1.2E-06 errors/bp Technique: M13mp2 forward mutation assay
T4 Kroutil LC1998 Nucleotide Substitution Rate 9.4E-07 errors/bp Technique: M13mp2 forward mutation assay
T4 Kroutil LC1998 Nucleotide Substitution Rate 3.3E-06 errors/bp Technique: M13mp2 opal codon reversion assay

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