ATP utilization by yeast replication factor C. I. ATP-mediated interaction with DNA and with proliferating cell nuclear antigen.
Eukaryotic replication factor C is the heteropentameric complex that loads the replication clamp proliferating cell nuclear antigen (PCNA) onto primed DNA. In this study we used a derivative, designated RFC, with a N-terminal truncation of the Rfc1 subunit removing a DNA-binding domain not required for clamp loading. Interactions of yeast RFC with PCNA and DNA were studied by surface plasmon resonance. Binding of RFC to PCNA was stimulated by either adenosine (3-thiotriphosphate) (ATPgammaS) or ATP. RFC bound only to primer-template DNA coated with the single-stranded DNA-binding protein RPA if ATPgammaS was also present. Binding occurred without dissociation of RPA. ATP did not stimulate binding of RFC to DNA, suggesting that hydrolysis of ATP dissociated DNA-bound RFC. However, when RFC and PCNA together were flowed across the DNA chip in the presence of ATP, a signal was observed suggesting loading of PCNA by RFC. With ATPgammaS present instead of ATP, long-lived response signals were observed indicative of loading complexes arrested on the DNA. A primer with a 3' single-stranded extension also allowed loading of PCNA; yet turnover of the reaction intermediates was dramatically slowed down. Filter binding experiments and analysis of proteins bound to DNA-magnetic beads confirmed the conclusions drawn from the surface plasmon resonance studies.
Gomes
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Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Burgers
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D051818:Replication Protein C; D029701:Saccharomyces cerevisiae Proteins; D020349:Surface Plasmon Resonance; D015003:Yeasts / Q000737:chemistry*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D018809:Proliferating Cell Nuclear Antigen; C087491:RFC2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D051818:Replication Protein C