An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex

Zhihao Zhuang, Anthony J. Berdis, Stephen Benkovic

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form in solution raises the possibility of other pathways for clamp loading. In this study, an alternative clamp loading sequence is evaluated in which gp44/62 first recognizes the DNA substrate and then sequesters the clamp from solution and loads it onto DNA. This pathway differs in terms of the initial formation of a gp44/62-DNA complex that is capable of loading gp45. In this work, we demonstrate ATP-dependent DNA binding by gp44/62. Among various DNA structures that were tested, gp44/62 binds specifically to primer-template DNA but not to single-stranded DNA or blunt-end duplex DNA. By tracing the dynamic clamp closing with pre-steady-state FRET measurements, we show that the clamp loader-DNA complex is functional in clamp loading. Furthermore, pre-steady-state ATP hydrolysis experiments suggest that 1 equiv of ATP is hydrolyzed when gp44/62 binds to DNA, and additional ATP hydrolysis is associated with the completion of the clamp loading process. We also investigated the detailed kinetics of binding of MANT-nucleotide to gp44/62 through stopped-flow FRET and demonstrated a conformational change as the result of ATP, but not ADP binding. The collective kinetic data allowed us to propose and evaluate a sequence of steps describing this alternative pathway for clamp loading and holoenzyme formation.

Original languageEnglish (US)
Pages (from-to)7976-7989
Number of pages14
JournalBiochemistry
Volume45
Issue number26
DOIs
StatePublished - Jul 4 2006

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Loaders
Clamping devices
DNA
Adenosine Triphosphate
Hydrolysis
Bacteriophage T4
Holoenzymes
DNA Primers
Single-Stranded DNA
Adenosine Diphosphate
Bacteriophages
Kinetics
Nucleotides

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Zhuang, Zhihao ; Berdis, Anthony J. ; Benkovic, Stephen. / An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex. In: Biochemistry. 2006 ; Vol. 45, No. 26. pp. 7976-7989.
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An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex. / Zhuang, Zhihao; Berdis, Anthony J.; Benkovic, Stephen.

In: Biochemistry, Vol. 45, No. 26, 04.07.2006, p. 7976-7989.

Research output: Contribution to journalArticle

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