Reconstitution of recombination-dependent DNA synthesis in herpes simplex virus 1

Amitabh V. Nimonkar, Paul E Boehmer

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

The repair of double-strand DNA breaks by homologous recombination is essential for the maintenance of genome stability. In herpes simplex virus 1, double-strand DNA breaks may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection. Double-strand DNA breaks are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization. We have reconstituted a system using purified proteins in which strand invasion is coupled with DNA synthesis. In this system, the viral single-strand DNA-binding protein promotes assimilation of single-stranded DNA into a homologous supercoiled plasmid, resulting in the formation of a displacement loop. The 3′ terminus of the invading DNA serves as a primer for long-chain DNA synthesis promoted by the viral DNA replication proteins, including the polymerase and helicase-primase. Efficient extension of the invading primer also requires a DNA-relaxing enzyme (eukaryotic topoisomerase I or DNA gyrase). The viral polymerase by itself is insufficient for DNA synthesis, and a DNA-relaxing enzyme cannot substitute for the viral helicase-primase. The viral single-strand DNA-binding protein, in addition to its role in the invasion process, is also required for long-chain DNA synthesis. Form X, a topologically distinct, positively supercoiled form of displacement-loop, does not serve as a template for DNA synthesis. These observations support a model in which recombination and replication contribute toward maintaining viral genomic stability by repairing double-strand breaks. They also account for the extensive branching observed during viral replication in vivo.

Original languageEnglish (US)
Pages (from-to)10201-10206
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number18
DOIs
StatePublished - Sep 2 2003
Externally publishedYes

Fingerprint

Human Herpesvirus 1
Genetic Recombination
Type I DNA Topoisomerase
Double-Stranded DNA Breaks
DNA
DNA Primase
Genomic Instability
DNA-Binding Proteins
DNA Gyrase
Single-Stranded DNA
Homologous Recombination
Viral DNA
Virus Diseases
DNA Replication
Proteins
Plasmids
Maintenance
Genome

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Reconstitution of recombination-dependent DNA synthesis in herpes simplex virus 1. / Nimonkar, Amitabh V.; Boehmer, Paul E.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, No. 18, 02.09.2003, p. 10201-10206.

Research output: Contribution to journalArticle

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