Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

Ravindra Kumar; Vishal C. Nashine; Padmaja P. Mishra; Stephen J. Benkovic; Tae Hee Lee

doi:10.1073/pnas.1014139107

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

Ravindra Kumar, Vishal C. Nashine, Padmaja P. Mishra, Stephen J. Benkovic, Tae Hee Lee

Research output: Contribution to journal › Article › peer-review

21 Scopus citations

Abstract

In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC • PCNA • DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC · PCNA · DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC · PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of polS only one species converts to the RFC · PCNA · DNA · polδ holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.

Original language	English (US)
Pages (from-to)	19736-19741
Number of pages	6
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	107
Issue number	46
DOIs	https://doi.org/10.1073/pnas.1014139107
State	Published - Nov 16 2010

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title = "Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies",

abstract = "In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC • PCNA • DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC · PCNA · DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC · PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of polS only one species converts to the RFC · PCNA · DNA · polδ holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.",

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AU - Nashine, Vishal C.

AU - Mishra, Padmaja P.

AU - Benkovic, Stephen J.

AU - Lee, Tae Hee

PY - 2010/11/16

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N2 - In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC • PCNA • DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC · PCNA · DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC · PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of polS only one species converts to the RFC · PCNA · DNA · polδ holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.

AB - In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC • PCNA • DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC · PCNA · DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC · PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of polS only one species converts to the RFC · PCNA · DNA · polδ holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.

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Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

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