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J Virol. 2010 Aug;84(15):7459-72. doi: 10.1128/JVI.01875-09. Epub 2010 May 5.

Processing of lagging-strand intermediates in vitro by herpes simplex virus type 1 DNA polymerase.

Author information

1
Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210, USA.

Abstract

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.

PMID:
20444887
PMCID:
PMC2897638
DOI:
10.1128/JVI.01875-09
[Indexed for MEDLINE]
Free PMC Article

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