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, and a processivity subunit, UL42, that, unlike other established processivity factors, binds DNA directly. We used gel retardation and filter-binding assays to investigate how UL42 affects the polymerase-DNA interaction. The Pol/UL42 heterodimer bound more tightly to DNA in a primer-template configuration than to single-stranded DNA (ssDNA), while Pol alone bound more tightly 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 approximately 15-fold relative to that of Pol. The affinity of Pol/UL42 for circular double-stranded DNA (dsDNA) was reduced drastically relative to that of UL42, but the affinity of Pol/UL42 for short primer-templates was increased modestly relative to that of UL42. Pol/UL42 associated with primer-template DNA approximately 2-fold faster than did Pol and dissociated approximately 10-fold more slowly, resulting in a half-life of 2 h and a subnanomolar Kd. Despite such stable binding, rapid-quench analysis revealed that the rates of elongation of Pol/UL42 and Pol were essentially the same, approximately 15 [corrected] nucleotides/s. Taken together, these studies indicate that (i) Pol/UL42 is more likely than its subunits to associate with DNA in a primer-template configuration rather than nonspecifically to either ssDNA or dsDNA, and (ii) UL42 reduces the rate of dissociation from primer-template DNA but not the rate of elongation. Two models of polymerase-DNA interactions during replication that may explain these findings are presented.
Weisshart
K
K
Department of Biological Chemistry and Molecular Pharmacology and Committee on Virology, Harvard Medical School, Boston Massachusetts 02115, USA.
Chow
C S
CS
Coen
D M
DM
D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D015684:Gene Products, pol / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D014764:Viral Proteins / Q000378:metabolism*
D015684:Gene Products, pol; D014764:Viral Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus; D008274:Magnesium