Oligomeric States of Bacteriophage T7 Gene 4 Primase/Helicase

Donald J. Crampton*, Melanie Ohi, Udi Qimron, Thomas Walz, Charles C. Richardson

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

52 Scopus citations

Abstract

Electron microscopic and crystallographic data have shown that the gene 4 primase/helicase encoded by bacteriophage T7 can form both hexamers and heptamers. After cross-linking with glutaraldehyde to stabilize the oligomeric protein, hexamers and heptamers can be distinguished either by negative stain electron microscopy or electrophoretic analysis using polyacrylamide gels. We find that hexamers predominate in the presence of either dTTP or β,γ-methylene dTTP whereas the ratio between hexamers and heptamers is nearly the converse in the presence of dTDP. When formed, heptamers are unable to efficiently bind either single-stranded DNA or double-stranded DNA. We postulate that a switch between heptamer to hexamer may provide a ring-opening mechanism for the single-stranded DNA binding pathway. Accordingly, we observe that in the presence of both nucleoside di- and triphosphates the gene 4 protein exists as a hexamer when bound to single-stranded DNA and as a mixture of heptamer and hexamer when not bound to single-stranded DNA. Furthermore, altering regions of the gene 4 protein postulated to be conformational switches for dTTP-dependent helicase activity leads to modulation of the heptamer to hexamer ratio.

Original languageEnglish
Pages (from-to)667-677
Number of pages11
JournalJournal of Molecular Biology
Volume360
Issue number3
DOIs
StatePublished - 14 Jul 2006
Externally publishedYes

Funding

FundersFunder number
National Institute of General Medical SciencesP01GM062580

    Keywords

    • DNA binding
    • helicase
    • heptamer
    • hexamer
    • phosphate sensor

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