There is amazing conservation in the requirements for and mechanism of DNA replication among highly diverse organisms. Herpes simplex virus type 1 (HSV-1) is an excellent model system for eukaryotic DNA replication since the virus encodes most of the proteins required for this essential process and can be manipulated genetically with greater ease than higher eukaryotes. DNA polymerases are central to the process of DNA replication. A general requirement of replicative DNA polymerases is that they copy the template genome with rapidity and reasonable fidelity. A major means by which they achieve the necessary rate required for genome duplication is the use of accessory proteins to increase their processivity. The HSV-1 DNA polymerase (pol) forms a stable and specific complex with an accessory factor, UL42. Like other pol accessory proteins, UL42 increases the processivity of its cognate pol, but differs in several important ways. Its lack of requirement for clamp loading proteins distinguishes it from the toroid sliding clamps, such as PCNA and E. coli pol III beta. Furthermore its intrinsic ability to bind to DNA is unique among all other known processivity factors, including those which don't require clamp loaders, such as thioredoxin, the processivity factor for T7 bacteriophage pol. The latter ability also presents an apparent paradox for known mechanisms of processivity, in that UL42 could also serve as a brake to elongation. The major long-term goal of the proposed studies is to elucidate the mechanism by which UL42 increases pol processivity, and the resulting impact this mechanism has on other properties of the pol, including parameters required for fidelity of DNA replication. A combination of biochemical, biophysical, and genetic approaches will be used to address four specific aims: 1) To determine the effect of reduced DNA binding by mutant UL42 proteins on rates of elongation and pol processivity using transient kinetic analysis and direct binding studies; 2) To determine the effect of processivity and proof-reading capability on the individual parameters which affect fidelity in vitro, including nucleotide selection, failure to extend mismatched termini, and excision of mismatched primer termini, using kinetic analysis to dissect these processes; 3) To determine the biological impact of changes in fidelity parameters (caused by changes in processivity) on the frequency and types of mutations which occur during origin (ori)- dependent DNA replication in vivo; 4) To determine the ability of the ori-binding protein, UL9, which interacts with UL42, to facilitate the assembly and/or processivity of pol/UL42 complexes on blocked synthetic primer/templates. Functional analogs of HSV-1 pol and UL42 are encoded by all human herpesviruses, including Kaposi sarcoma-associated virus (HHV-8), Epstein Barr virus, and human cytomegalovirus, all of which are significant human pathogens, particularly for cancer and immuno- suppressed patients. It is important to understand an the mechanism of UL42 action since disruption of the pol/UL42 complex has been proposed for development of antivirals.
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