Skip to main content
Journal cover image

Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly.

Publication ,  Journal Article
Soumillion, P; Sexton, DJ; Benkovic, SJ
Published in: Biochemistry
February 17, 1998

Clamp proteins confer processivity to the DNA polymerase during DNA replication. These oligomeric proteins are loaded onto DNA by clamp loader protein complexes in an ATP-dependent manner. The mechanism by which the trimeric bacteriophage T4 clamp protein (the 45 protein) loads and dissociates from DNA was investigated as a function of its intersubunit protein-protein interactions. These interactions were continuously monitored using a fluorescence resonance energy transfer (FRET) based assay. A cysteine mutant of the 45 protein was constructed to facilitate site-specific incorporation of a fluorescent probe at the subunit interface. This site was chosen such that FRET was observed between the introduced fluorescent probe and a tryptophan residue located on the opposing subunit. By use of this fluorescently labeled 45 protein, it was possible to obtain an estimate of an apparent trimer dissociation constant from either a cooperative (0.08 +/- 0.04 microM2 at 25 degrees C) or a noncooperative (0.51 microM and 0.17 microM at 25 degrees C) model. Upon mixing the fluorescently labeled 45 protein with a 45 protein containing 4-fluorotryptophan, a nonfluorescent tryptophan analogue, subunit exchange between the two variants of the 45 protein was observed according to a reduction in intersubunit FRET. Subunit exchange rate constants measured in the presence or absence of the clamp loader (44/62 complex), the polymerase (43 protein), and/or a primer template DNA substrate demonstrate (a) that the 45 protein is not loaded onto DNA by subunit exchange and (b) that the disassembly dissociation of a stalled holoenzyme from DNA is dictated by 45 protein subunit dissociation.

Duke Scholars

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

February 17, 1998

Volume

37

Issue

7

Start / End Page

1819 / 1827

Location

United States

Related Subject Headings

  • Viral Proteins
  • Valine
  • Tryptophan
  • Trans-Activators
  • Spectrometry, Fluorescence
  • Naphthalenesulfonates
  • Mutagenesis, Site-Directed
  • Kinetics
  • DNA-Directed DNA Polymerase
  • Cysteine
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Soumillion, P., Sexton, D. J., & Benkovic, S. J. (1998). Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly. Biochemistry, 37(7), 1819–1827. https://doi.org/10.1021/bi972526a
Soumillion, P., D. J. Sexton, and S. J. Benkovic. “Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly.Biochemistry 37, no. 7 (February 17, 1998): 1819–27. https://doi.org/10.1021/bi972526a.
Soumillion P, Sexton DJ, Benkovic SJ. Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly. Biochemistry. 1998 Feb 17;37(7):1819–27.
Soumillion, P., et al. “Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly.Biochemistry, vol. 37, no. 7, Feb. 1998, pp. 1819–27. Pubmed, doi:10.1021/bi972526a.
Soumillion P, Sexton DJ, Benkovic SJ. Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly. Biochemistry. 1998 Feb 17;37(7):1819–1827.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

February 17, 1998

Volume

37

Issue

7

Start / End Page

1819 / 1827

Location

United States

Related Subject Headings

  • Viral Proteins
  • Valine
  • Tryptophan
  • Trans-Activators
  • Spectrometry, Fluorescence
  • Naphthalenesulfonates
  • Mutagenesis, Site-Directed
  • Kinetics
  • DNA-Directed DNA Polymerase
  • Cysteine