Functional interaction between the herpes simplex-1 DNA polymerase and UL42 protein.
The herpes simplex virus 1 (HSV-1) UL42 protein, one of seven herpes-encoded polypeptides that are required for the replication of the HSV-1 genome, is found in a 1:1 complex with the HSV-1 DNA polymerase (Crute, J. J., and Lehman, I. R. (1989) J. Biol. Chem. 264, 19266-19270). To obtain herpes DNA polymerase free of UL42 protein, we have cloned and overexpressed the Pol gene in a recombinant baculovirus vector and purified the recombinant DNA polymerase to near homogeneity. Replication of singly primed M13mp18 single-stranded DNA by the recombinant enzyme in the presence of the herpes encoded single-stranded DNA-binding protein ICP8 yields in addition to some full-length product a distribution of intermediate length products by a quasi-processive mode of deoxynucleotide polymerization. Addition of the purified UL42 protein results in completely processive polymerization and the generation of full-length products. Similar processivity is observed with the HSV-1 DNA polymerase purified from herpes-infected Vero cells. Processive DNA replication by the DNA polymerase isolated from HSV-1-infected Vero cells or the recombinant DNA polymerase-UL42 protein complex requires that the single-stranded DNA be coated with saturating levels of ICP8. ICP8 which binds single-stranded DNA in a highly cooperative manner is presumably required to melt out regions of secondary structure in the single-stranded DNA template, thereby potentiating the processivity enhancing action of the UL42 protein.
Department of Biochemistry, Beckman Center, Stanford University School of Medicine, California 94305-5307.
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Hernandez
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Lehman
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism* / Q000494:pharmacology
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D014764:Viral Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus