Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the a-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain a-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that a-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an a-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between a-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in a-like DNA polymerases.DNA polymerases are central to the replication of genetic material. Much of our information regarding DNA polymerases has come from detailed structural, enzymological, and genetic studies of Escherichia coli DNA polymerase I (Pol I) and its relative, T7 DNA polymerase (33, 61). However, most eukaryotic cellular and viral replicative DNA polymerases and certain bacterial and bacteriophage DNA polymerases are members of the (x-like polymerase family and share only very limited sequence similarity with Pol I (1,39,45,60,68). A fundamental question is: Do these enzymes carry out their functions similarly to or differently from Pol I?DNA polymerases usually contain both polymerase and exonucleolytic activities either on a single polypeptide or as separate subunits. The 3'-5' exonuclease activities associated with polymerases frequently perform proofreading functions, thereby reducing replication errors and mutation rates. A key feature of Pol I is that its polymerase and 3'-5' exonuclease activities reside on a single polypeptide (37) as separate domains with the active sites about 3 nm apart (47). This physical separation translates into functional independence, as revealed by analysis of mutations, including fairly gross deletions, of Pol I and T7 DNA polymerase that inactivate 3'-5' exonuclease activity without major deleterious effects on polymerase activity and processivity (11,12,19,61).Interestingly, a-like DNA polymerases share sequence similarities with active sites in the 3'-5' exonuclease domain of Pol I (2, 39, 45, 60). Bernad et al. (2) have termed these segments Exo I, Exo II, and Exo III (Fig...