Herpes Simplex Virus Processivity Factor UL42 Imparts Increased DNA-Binding Specificity to the Viral DNA Polymerase and Decreased Dissociation from Primer-Template without Reducing the Elongation Rate

During DNA replication, long-chain DNA synthesis on the leading strand requires processivity factors to overcome the tendency of DNA polymerase to dissociate from the template after each catalytic step. In most of the organisms studied to date, these processivity factors interact with the catalytic subunit of polymerase and with DNA to increase the time of association of the holoenzyme with the DNA template. The best-characterized processivity factors are the so-called “sliding clamps,” which include the Escherichia coli β subunit of DNA polymerase III, bacteriophage T4 gp45, and eukaryotic PCNA. Biochemical and crystallographic studies have shown that these factors do not bind directly to DNA but, rather, form multimeric rings around DNA, which permits them to slide along the template (15, 19, 30, 31, 47, 58). Under physiological conditions, the association of a sliding clamp with DNA and its cognate polymerase requires auxiliary proteins that serve as “clamp loaders” (27, 32, 33, 40, 50) and occurs at the primer-template junction (reviewed in reference 20, 27, 44, and 45). A second type of processivity factor is exemplified by thioredoxin, which, unlike the sliding clamps, associates with T7 DNA polymerase in the absence of other protein factors or ATP to form a heterodimer (22, 24, 39, 48). However, like sliding clamps, thioredoxin does not bind DNA directly (39). Thus, during DNA synthesis, the interactions of the sliding clamps and thioredoxin with DNA and their translocation appear to be passive and dependent on their association with their cognate polymerases. For herpes simplex virus (HSV), the details of how processive DNA synthesis occurs have yet to be resolved. The replicative DNA polymerase consists of a heterodimer of two proteins (6, 14). One of these proteins is the catalytic subunit, Pol (also known as UL30), whose intrinsic activities include a 5′-3′ polymerase and a 3′-5′ exonuclease (11, 16, 17, 29, 35, 42, 56). To carry out these functions, Pol has double-stranded DNA (dsDNA)- and single-stranded DNA (ssDNA)-binding activities that also can be found within individual proteolytic fragments of the enzyme (56). Moreover, DNA binding causes a conformational change in Pol (57). However, this DNA binding is not sufficient for highly processive DNA synthesis (14, 21). The second HSV polymerase subunit is UL42, which is a processivity factor (14). It differs from other established processivity factors in that it binds directly and stably to dsDNA, albeit without sequence specificity (36, 43). Unlike sliding clamps, the association of Pol and UL42 does not require additional factors and can occur in the absence of DNA. Both the DNA-binding and Pol-binding activities of UL42 appear to be required for its function as a processivity factor, since mutations that specifically affect either of these activities severely reduce long-chain DNA synthesis and in vivo replication (3, 10). Moreover, the affinity of Pol/UL42 for a hairpin primer-template is greater than that of Pol alone, and the footprint on the dsDNA region of this template is extended when UL42 is present (13). Thus, the available data support the hypothesis (14) that UL42 functions as a tether between Pol and DNA and that its DNA-binding activity is crucial for its function as a processivity factor. However, many of the specifics regarding how UL42 affects the interaction of polymerase with DNA are not known, nor is it known whether UL42, which has high affinity for DNA, affects the movement of the polymerase along the template. In this study, we tested three hypotheses: (i) that UL42 increases the specificity of the holoenzyme for a primer-template configuration, (ii) that UL42 limits the rate of dissociation of the holoenzyme from the primer-template, and (iii) that the increase in processivity conferred by UL42 is achieved at the expense of a decrease in the rate of elongation, i.e., that the polymerase sacrifices speed for distance. To examine these hypotheses, bandshift and competition assays were used to determine the binding affinities of Pol, UL42, and the Pol/UL42 heterodimer on different templates in the presence or absence of magnesium. These assays allowed a comparison of the binding preferences of these proteins to different configurations of DNA. The association and dissociation rates of Pol and Pol/UL42 on DNA in a primer-template configuration were determined. Finally, the rates of elongation of Pol and Pol/UL42 were compared by using a rapid-quench technique. The results indicate that UL42 both increases the specificity of polymerase binding to primer-template DNA and decreases the rate of dissociation from primer-template DNA. However, despite the increased binding to DNA, UL42 does not retard the rate of elongation of polymerase.