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 sh owed that exonuclease-deficient T4 DNA polymerase, alone or with clamp loaders gp44/gp62 and polymerase clamp gp45, displays decreased bindi ng affinity for incorrect as compared to correct dNTPs and a deceased k(cat) for misinsertion as compared to correct insertion. Kinetic cons tants were similar with and without accessory proteins, indicating tha t accessory proteins had little effect on misinsertion. They also had little effect on the K-m value for extension of a T.T mismatch. Howeve r, the k(cat), value for T.T mismatch extension was fivefold higher in the presence of the clamp loader and clamp proteins. Thus, in the abs ence of proofreading, these accessory proteins may promote stable misi ncorporation. The kinetic analysis is supported by error rate determin ations during gap-filling synthesis, which require both misinsertion a nd mispair extension. For some mispairs, the accuracy of exonuclease-d eficient polymerase alone is similar to that in the presence of clamp loader, clamp and single-stranded DNA binding protein (gpp32). However , exonuclease-deficient holoenzyme complex is actually less accurate t han the polymerase alone for some base substitutions. We suggest that gp45 promotes extension of mismatches by tethering the polymerase to D NA, a process that may be relevant to replication past lesions or othe r 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 a rise 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 all ow misalignment but remains tethered to the template through interacti ons with the clamp. Finally, the T4 replication accessory proteins red uced by greater than or equal to 10-fold the rate at which exonuclease -deficient T4 DNA polymerase generated deletions of larger numbers of nucleotides, indicating that these proteins influence replication fide lity for other than single base mutations.