Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies.
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 polδ 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.
Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
Ravindra
R
Kumar
Vishal C
VC
Nashine
Padmaja P
PP
Mishra
Stephen J
SJ
Benkovic
Tae-Hee
TH
Lee
D000255:Adenosine Triphosphate / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000378:metabolism; D051818:Replication Protein C / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D004271:DNA, Fungal; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; C084903:RFC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D051818:Replication Protein C