Rapid assembly of the bacteriophage T4 core replication complex on a linear primer/template construct

Barbara Fenn Kaboord, Stephen Benkovic

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

DNA synthesis on a primed DNA substrate by bacteriophage T4 requires the assembly of a core replication complex consisting of the T4 DNA polymerase, a single-stranded binding protein (32 protein), and the accessory proteins 44/62 and 45. In this paper, we demonstrate the successful assembly of this core complex on a short linear primer/template system at levels of accessory proteins equivalent to the concentration of primer 3′ ends. The key to this assembly is the presence of streptavidin molecules bound at each end of the DNA substrate via biotin moieties incorporated into the template strand. Streptavidin serves to block the ends of the primer/template, thus preventing translocation of the accessory proteins away from the site of assembly and their subsequent dissociation from the ends of the primer/template. Complex assembly on this substrate requires ATP and the presence of both the 44/62 and 45 proteins. The time required for assembly of a full enzyme equivalent of complex in our system is ≈2 s.

Original languageEnglish (US)
Pages (from-to)10881-10885
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number22
DOIs
StatePublished - Nov 15 1993

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Bacteriophage T4
Streptavidin
DNA
Proteins
Protein Transport
DNA-Directed DNA Polymerase
Biotin
Carrier Proteins
Adenosine Triphosphate
Enzymes

All Science Journal Classification (ASJC) codes

  • General

Cite this

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Rapid assembly of the bacteriophage T4 core replication complex on a linear primer/template construct. / Kaboord, Barbara Fenn; Benkovic, Stephen.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, No. 22, 15.11.1993, p. 10881-10885.

Research output: Contribution to journalArticle

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