Stepwise assembly of the human replicative polymerase holoenzyme

Mark Hedglin, Senthil K. Perumal, Zhenxin Hu, Stephen Benkovic

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (polδ), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication.

Original languageEnglish (US)
Article numbere00278
JournaleLife
Volume2013
Issue number2
DOIs
StatePublished - Apr 2 2013

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Holoenzymes
Proliferating Cell Nuclear Antigen
Clamping devices
DNA
Loaders
Recycling
DNA-Directed DNA Polymerase
Unloading
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Medicine(all)
  • Neuroscience(all)

Cite this

Hedglin, Mark ; Perumal, Senthil K. ; Hu, Zhenxin ; Benkovic, Stephen. / Stepwise assembly of the human replicative polymerase holoenzyme. In: eLife. 2013 ; Vol. 2013, No. 2.
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Stepwise assembly of the human replicative polymerase holoenzyme. / Hedglin, Mark; Perumal, Senthil K.; Hu, Zhenxin; Benkovic, Stephen.

In: eLife, Vol. 2013, No. 2, e00278, 02.04.2013.

Research output: Contribution to journalArticle

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