Processing of lagging-strand intermediates in vitro by herpes simplex virus type 1 DNA polymerase.
The processing of lagging-strand intermediates has not been demonstrated in vitro for herpes simplex virus type 1 (HSV-1). Human flap endonuclease-1 (Fen-1) was examined for its ability to produce ligatable products with model lagging-strand intermediates in the presence of the wild-type or exonuclease-deficient (exo(-)) HSV-1 DNA polymerase (pol). Primer/templates were composed of a minicircle single-stranded DNA template annealed to primers that contained 5' DNA flaps or 5' annealed DNA or RNA sequences. Gapped DNA primer/templates were extended but not significantly strand displaced by the wild-type HSV-1 pol, although significant strand displacement was observed with exo(-) HSV-1 pol. Nevertheless, the incubation of primer/templates containing 5' flaps with either wild-type or exo(-) HSV-1 pol and Fen-1 led to the efficient production of nicks that could be sealed with DNA ligase I. Both polymerases stimulated the nick translation activity of Fen-1 on DNA- or RNA-containing primer/templates, indicating that the activities were coordinated. Further evidence for Fen-1 involvement in HSV-1 DNA synthesis is suggested by the ability of a transiently expressed green fluorescent protein fusion with Fen-1 to accumulate in viral DNA replication compartments in infected cells and by the ability of endogenous Fen-1 to coimmunoprecipitate with an essential viral DNA replication protein in HSV-1-infected cells.
Zhu
Yali
Y
Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210, USA.
Wu
Zetang
Z
Cardoso
M Cristina
MC
Parris
Deborah S
DS
D001483:Base Sequence; D047369:Chromatin Immunoprecipitation; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D045585:Flap Endonucleases / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C043417:DNA polymerase, Simplexvirus; C468317:FEN1 protein, human
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