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Residues in the central β-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding

Journal Article


Abstract


  • The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic β-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the β-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468 are essential for both DNA unwinding and DNA synthesis mediated by T7 DNA polymerase during leading-strand DNA synthesis. Gp4-K467A, gp4-K471A, and gp4-K473A form fewer hexamers than heptamers compared to wild-type helicase and alone are deficient in DNA unwinding. However, they complement for the growth of T7 bacteriophage lacking gene 4. Single-molecule studies show that these three altered helicases support rates of leading-strand DNA synthesis comparable to that observed with wild-type gp4. Gp4-K467A, devoid of unwinding activity alone, supports leading-strand synthesis in the presence of T7 DNA polymerase. We propose that DNA polymerase limits the backward movement of the helicase during unwinding as well as assisting the forward movement necessary for strand separation.

Authors


  •   Satapathy, Ajit K. (external author)
  •   Kochaniak, Anna B. (external author)
  •   Mukherjee, Sourav (external author)
  •   Crampton, Donald J. (external author)
  •   van Oijen, Antoine M.
  •   Richardson, Charles C. (external author)

Publication Date


  • 2010

Citation


  • Satapathy, A. K., Kochaniak, A. B., Mukherjee, S., Crampton, D. J., van Oijen, A. M. & Richardson, C. C. (2010). Residues in the central β-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding. Proceedings of the National Academy of Sciences of USA, 107 (15), 6782-6787.

Scopus Eid


  • 2-s2.0-77951036168

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2178

Number Of Pages


  • 5

Start Page


  • 6782

End Page


  • 6787

Volume


  • 107

Issue


  • 15

Abstract


  • The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic β-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the β-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468 are essential for both DNA unwinding and DNA synthesis mediated by T7 DNA polymerase during leading-strand DNA synthesis. Gp4-K467A, gp4-K471A, and gp4-K473A form fewer hexamers than heptamers compared to wild-type helicase and alone are deficient in DNA unwinding. However, they complement for the growth of T7 bacteriophage lacking gene 4. Single-molecule studies show that these three altered helicases support rates of leading-strand DNA synthesis comparable to that observed with wild-type gp4. Gp4-K467A, devoid of unwinding activity alone, supports leading-strand synthesis in the presence of T7 DNA polymerase. We propose that DNA polymerase limits the backward movement of the helicase during unwinding as well as assisting the forward movement necessary for strand separation.

Authors


  •   Satapathy, Ajit K. (external author)
  •   Kochaniak, Anna B. (external author)
  •   Mukherjee, Sourav (external author)
  •   Crampton, Donald J. (external author)
  •   van Oijen, Antoine M.
  •   Richardson, Charles C. (external author)

Publication Date


  • 2010

Citation


  • Satapathy, A. K., Kochaniak, A. B., Mukherjee, S., Crampton, D. J., van Oijen, A. M. & Richardson, C. C. (2010). Residues in the central β-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding. Proceedings of the National Academy of Sciences of USA, 107 (15), 6782-6787.

Scopus Eid


  • 2-s2.0-77951036168

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2178

Number Of Pages


  • 5

Start Page


  • 6782

End Page


  • 6787

Volume


  • 107

Issue


  • 15