The UL8 Subunit of the Heterotrimeric Herpes Simplex Virus Type 1 Helicase-Primase Is Required for the Unwinding of Single Strand DNA-binding Protein (ICP8)-coated DNA Substrates

Maria Falkenberg,David A Bushnell,Per Elias,I.R Lehman

doi:10.1074/jbc.272.36.22766

Abstract

The Herpes simplex virus type 1 primosome consists of three subunits that are the products of the UL5, UL8, and UL52 genes. The heterotrimeric enzyme has DNA-dependent ATPase, helicase, and primase activities. Earlier studies show that a subassembly consisting of the UL5 and UL52 gene products was indistinguishable from the heterotrimeric enzyme in its helicase and primase activities. We demonstrate here that the UL8 protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates (>30 nucleotides) with a single-stranded DNA loading site fully coated with the virus-encoded single strand DNA binding protein, ICP8. The Escherichia coli single strand DNA binding protein cannot substitute for ICP8, suggesting a specific physical interaction between ICP8 and the UL8 protein. Surface plasmon resonance measurements demonstrated an interaction between ICP8 and the UL5/52/8 heterotrimer but not with the UL5/52 subassembly or the UL8 protein alone. At a subsaturating level of ICP8, the UL5/52 subassembly does show helicase activity, suggesting that the subassembly can bind to single-stranded DNA but not to ICP8-coated DNA.

Highlights

Herpes simplex virus type 1 (HSV-1)1 encodes seven proteins that are essential for the replication of its genome [1]
The UL8 gene product is essential for HSV-1 DNA replication in vivo, a subassembly consisting of only the 99-kDa UL5 and 114-kDa UL52 gene products was found to be indistinguishable in its DNA-dependent ATPase, DNA helicase, and DNA primase activities from the heterotrimeric enzyme [13, 14]
In the present study we have found that with duplex DNA substrates Ͼ30 nucleotides in length containing a single-stranded loading site fully coated with ICP8, helicase activity shows an almost complete dependence on the UL8 subunit

Summary

Introduction

Herpes simplex virus type 1 (HSV-1) encodes seven proteins that are essential for the replication of its genome [1]. These include an origin binding protein [2, 3], a single strand DNA binding protein [4], a DNA polymerase [5] with its associated processivity factor (6 – 8), and a heterotrimeric primosome. It was found that the UL8 protein can stimulate the DNA-dependent ATPase, helicase, and primase activities of the UL5/52 heterodimer in the presence of the HSV-1-encoded single strand DNA-binding protein, ICP8 [18]. The helicase activity of the HSV-1-encoded primosome appears to be modulated by ICP8

Methods

Results

Conclusion