Herpes simplex virus is a useful model for studying the mechanisms involved in DNA replication in eukaryotic cells. Our current efforts are directed toward studying this process with purified proteins. Seven viral genes are both necessary and sufficient to carry out authentic origin-dependent DNA replication. Ongoing biochemical analyses in several laboratories support the idea that the products of these seven genes all participate directly in viral DNA synthesis. We are currently using both biochemical and molecular genetic approaches to understand the function of these polypeptides in detail. The HSV DNA polymerase purified from infected HeLa cells consists of a stable complex of two polypeptides: UL30, the catalytic subunit, and UL42, and accessory subunit. Several lines of evidence support the view UL42 increases the efficiency of the DNA polymerase by increasing its processivity. Using a novel synthetic model primer-template, we have characterized the interaction of the HSV DNA polymerase with its nucleic acid substrate. Our results suggest that UL42 increases the processivity of DNA polymerase by acting as a sliding clamp, reducing the probability that the polymerase will dissociate from the elongating DNA chain after each cycle of catalysis. The UL5, UL8, and UL52 polypeptides form a three protein complex that has both helicis and primase activities. Purification of the isolated subunits and subcomplex associations of subunits suggests that UL5 and UL52 act together to catalyze both helicase and primase activities. On the basis of sequence motifs, we presume that UL5 has the active site for helicase function, but UL5 is no active as a helicase in the absence of UL52. We propose that UL52 has the active site for primase function, and are trying to obtain direct support for this view. Although UL8 is required for neither helicase nor primase activities, it is required for the efficient utilization of primers by DNA polymerase in a model system for lagging strand synthesis. We suggest that the role of UL8 is to stabilize the association of nascent primers with the template, increasing the probability that primers are elongated by DNA polymerase. Work is underway to test this model.
| Vashee, S; Simancek, P; Challberg, M D et al. (2001) Assembly of the human origin recognition complex. J Biol Chem 276:26666-73 |
