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 to analyzed a number of mutants in conserved sequence motifs in the helicase subunit of the helicase/ lprimase. Our results have identified a number of mutants that are able to both hyudrolyze ATP and bind to single-and double standed DNA, but cannot couple these two activities in a way that leads to DNA unwinding. Further analysis of these mutants should lead to new insights into the mechanism of helicases, a key enzyme activity not only in DNA replication but also a number of other cell processes, including recombination, DNA repair, transcription and translation. Second, 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 two subunits of HSV-1 origin and one subunit (the helicase subunit) of HSV-2 origin. We have over-expressed and purified this heterotypic enzyme. Biochemical analyses suggest that the heterotypic enzyme has greatly reduced primase activity relative to the HSV-1 homotypic enzyme. This result suggests that interactions between the subunits are critical for full enzyme activity and small changes such as the 5% difference between the HSV-1 and HSV-2 homologs can have a profound effect on the interaction. Moreover, the results also suggest that the details of DNA replication must be substantially different in neurons than in other cells, since the effect of this quantitative primase deficiency is only apparent in neurons.
| Vashee, S; Simancek, P; Challberg, M D et al. (2001) Assembly of the human origin recognition complex. J Biol Chem 276:26666-73 |
