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

Herpes simplex virus DNA polymerase consists of a catalytic subunit, Pol, a nd a processivity subunit, UL42, that, unlike other established processivit y factors, binds DNA directly, We used gel retardation and filter-binding a ssays to investigate how UL42 affects the polymerase-DNA interaction, The P ol/UL42 heterodimer bound more tightly to DNA in a primer-template configur ation than to single-stranded DNA (ssDNA), while Pol alone bound more tight ly to ssDNA than to DNA in a primer-template configuration, The affinity of Pol/UL42 for ssDNA was reduced severalfold relative to that of Pol, while the affinity of Pol/UL42 for primer-template DNA was increased similar to 1 5-fold relative to that of Pol, The affinity of Pol/UL42 for circular doubl e-stranded DNA (dsDNA) was reduced drastically relative to that of UL42, bu t the affinity of Pol/UL42 for short primer-templates was increased modestl y relative to that of UL42, Pol/UL42 associated with primer-template DNA si milar to 2-fold faster than did Pol and dissociated similar to 10-fold more slowly, resulting in a half-life of 2 h and a subnanomolar K-d. Despite su ch stable binding, rapid-quench analysis revealed that the rates of elongat ion of Pol/UL42 and Pol were essentially the same, similar to 30 nucleotide s/s. Taken together, these studies indicate that (i) Pol/UL42 is more likel y than its subunits to associate with DNA in a primer-template configuratio n rather than nonspecifically to either ssDNA or dsDNA, and (ii) UL42 reduc es the rate of dissociation from primer-template DNA but not the rate of el ongation. Two models of polymerase-DNA interactions during replication that may explain these findings are presented.