The primary goal of our research is to understand the mechanisms involved in DNA replication in eukaryotic cells. We have chosen to study these processes using the herpesviruses as a model, since this affords the opportunity to not only gain new insights into the process of DNA replication itself, but also to learn more about the biology of an important human pathogen. In the past year, we have made the following progress. First, we have begun to define the role of the essential replication protein UL8, which is a component of a helicase primase complex but has no known enzymatic activity. This protein appears to stimulate the activity of the other two subunits, either by changing their conformation or by altering the interaction of the complex with DNA. Second, we have begun to analyze a number of mutants in conserved sequence motifs in the helicase subunit of the helicase/lprimase. This analysis will help to define the catalytic function of these sequence motifs and should lead to new insights into the mechanism of helicases. Third, we have analyzed an HSV-1/HSV-2 intertypic mutant of HSV which has previously been reported to have a specific defect in DNA replication in cells of neuronal origin, but carries out DNA replication normally in other cell types. We have found that the neuronal specific defect can be traced to the fact that the mutant forms a heterotypic helicase primase complex, with the UL52 and UL8 subunits of HSV-1 origin and the UL5 subunit of HSV-2 origin. We have overexpressed and purified this heterotypic enzyme. Preliminary biochemical analyses suggest that the heterotypic enzyme has greatly reduced primase activity relative to the HSV-1 homotypic enzyme. This result suggets that 1) host cell primase may be able to compensate for the lack of virus-encoded primase; and 2) the neuronal cell specific defect in DNA replication may be due to the fact that neuronal cells, which do not carry out replicative DNA synthesis, lack adequate levels of primase.
| Vashee, S; Simancek, P; Challberg, M D et al. (2001) Assembly of the human origin recognition complex. J Biol Chem 276:26666-73 |
