Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3' -S 5' exonuclease activity. A mutant DNA polymerase with very little (<1%) 3' -*5' exonuclease activity was generated. In vivo, the 3' -*5' exonuclease-deficient DNA polymerase produced the highest level ofspontaneous mutation observed in T4, 500-to 1800-fold above that of wild type. The large reduction in 3' -* 5' exonuclease activity appears to be due to two amino acid substitutions: clease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.Bacteriophage T4 DNA polymerase is one of the best experimental systems for studying the role of DNA polymerase exonucleolytic proofreading in enhancing DNA replication fidelity (1-10). Wild-type T4 DNA polymerase has a potent 3' 5' exonuclease activity (11), which is important for accurate DNA replication. Mutant DNA polymerases with reduced 3' -* 5' exonuclease activity produce more DNA replication errors (mutator phenotype), whereas mutants with elevated 3' 5' exonuclease activity, relative to polymerizing activity, increase DNA replication accuracy (antimutator phenotype) (3). The mutator phenotype was used to select mutant DNA polymerases with reduced 3' -* 5' exonuclease activity; amino acid substitutions in the mutants were clustered'between T4 DNA polymerase residues 255 and 363 (8)(9)(10)12). Although amino acid changes within this region decreased 3' -* 5' exonuclease activity, the' mutant DNA polymerases still retained significant residual proofreading activity, which suggests that these particular residues do not function catalytically.In the case of Escherichia coli DNA polymerase I (pol I), residues essential for 3' 5' exonuclease activity have been identified. They include four metal ion binding residues; The hypothesis drawn from these sequence comparisons was that many eukaryotic, viral, and bacteriophage DNA polymerases have a conserved 3' -* 5' exonuclease domain similar to that of E. coli pol 1. T4 genetic studies are consistent with this proposal because mutations that reduce 3' -* 5' exonuclease activity are located near the proposed conserved metal ion binding residues. The hypothesis was tested directly in phage 429'DNA polymerase by substituting alanine residues for proposed conserved metal ion binding residues, which resulted in a 1000-fold reduction in 3' -* 5' exonuclease activity without affecting polymerization activity (23). The 429 DNA polymerase result compares favorably with the 105-fold reduction observed when alanine residues were substituted for E. coli pol I residues .We present here in vitro mutagenesis and genetic studies that were designed to test if proposed T4 DNA' polymerase metal ion binding residues, identified on the basis of sequence similarities to those of E. coli pol l (8, 23), are required for 3' 5' exonuclease activity. In 'contrast to the 429 DNA polymerase'studies, alanine subs...

Using indirect immunofluorescence, well-characterized monoclonal and polyclonal antibodies, and temperature-sensitive (ts) mutants of herpes simplex virus type 1, we demonstrated that the 65-kilodalton DNAbinding protein (65KDBP), the major DNA-binding protein (infected cel polypeptide 8 [ICP8]), and the viral DNA polymerase (Pol) colocalize to replication compartments in the nuclei of infected cells under conditions which permit viral DNA synthesis. When viral DNA synthesis was blocked by incubation of the wild-type virus with phosphonoacetic acid, the 65KDBP, Pol, and ICP8 failed to localize to replication compartments. Instead, ICP8 accumulated nearly exclusively to prereplication sites, while the 65KDBP was only diffusely localized within the nuclei. Although some of the Pol accumulated in prereplication sites occupied by ICP8 in the presence of phosphonoacetic acid, a significant amount of Pol also was distributed throughout the nuclei. Examination by double-labeling immunofluorescence of DNA-ts mutant virus-infected cells revealed that the 65KDBP also did not colocalize with ICP8 to prereplication sites at temperatures nonpermissive for virus replication. These results are in disagreement with the hypothesis that ICP8 is the major organizational protein responsible for attracting other replication proteins to prereplication sites in preparation for viral DNA synthesis (A. de Bruyn Kops and D. M. Knipe, Cell 55:857-868, 1988), and they suggest that other viral proteins, perhaps in addition to ICP8, or replication fork progression per se are required to organize the 65KDBP.

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The 65-kilodalton DNA-binding protein (65KDBp) of herpes simplex virus type 1, encoded by gene UL42, is required for herpes simplex virus origin-dependent DNA replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). We found by indirect immunofluorescence with monoclonal antibody to 65KDBP that the protein was first detectable at 3 h postinfection. It localized first to the inner periphery of the nucleus, but accumulated in large globular compartments within the nucleus by 6 h postinfection in a pattern similar to that displayed by the major DNA-binding protein ICP8. Immune electron microscopy revealed that 65KDBP was associated with the marginated heterochromatin at the early times, but migrated further into the nucleus at late times when the only discernible areas devoid of 65KDBP were the nucleoli and heterochromatin. The 65KDBP gene is a member of the 0i kinetic class as determined by the ability of the mRNA to be expressed at significant levels even in the absence of viral DNA synthesis. Furthermore, in the presence or absence of the DNA polymerase inhibitor phosphonoacetic acid, the patterns of accumulation of protein as well as mRNA were virtually indistinguishable from those displayed by the model 0 genes encoding ICP8 and thymidine kinase. Nuclear run-on experiments demonstrated that maximum rates of

Three T4 DNA polymerase accessory proteins (44P/62P and 45P) stimulate the polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of T4 DNA polymerase (43P) on long, double-stranded DNA substrates. The 44P/62P "clamp loader" facilitates the binding of 45P, the "sliding clamp", to DNA that is primed for replication. Using a series of truncated 43P mutants, we identified a region at the extreme carboxy terminus of the DNA polymerase that is required for its interaction with accessory proteins. Truncation mutants of 43P lacking the carboxy-terminal 3, 6, or 11 residues retained full pol and exo activity on short synthetic primer-templates. However, the ability of the accessory proteins to enhance these activities on long double-stranded DNA templates was drastically reduced, and the extent of the reduction in activity was greater as more residues were deleted. One of the truncation mutants (N881), which had 17 residues removed from the carboxy terminus, showed reduced binding affinity and diminished pol activity but enhanced exo activity upon incubation with a small primer-template. The exo activity of the N881 mutant, on short, single-stranded DNA was unchanged, however, compared to the wild-type enzyme. These results are consistent with inferences drawn from the crystal structure of a DNA polymerase from a related T-even phage, RB69, where the carboxy-terminal 12 residues (equivalent to the 11 residues of 43P from phage T4) protrude from the thumb domain and are free to interact with complementary surfaces of the accessory proteins. The structural integrity of the thumb region in the N881 mutant is probably perturbed and could account for its reduced binding affinity and pol activity when incubated with short, double-stranded DNA substrates.