ATP utilization by yeast replication factor C: II. Multiple stepwise ATP binding events are required to load proliferating cell nuclear antigen onto primed DNA

Xavier V. Gomes, Sonja L. Gary Schmidt, Peter M.J. Burgers

Research output: Contribution to journalArticlepeer-review

85 Scopus citations

Abstract

Binding of adenosine (3-thiotriphosphate) (ATPγS), a nonhydrolyzable analog of ATP, to replication factor C with a N-terminal truncation (Δ2-273) of the Rfc1 subunit (RFC) was studied by filter binding. RFC alone bound 1.8 ATPγS molecules. However, when either PCNA or primer-template DNA were also present 2.6 or 2.7 ATPγS molecules, respectively, were bound. When both PCNA and DNA were present 3.6 ATPγS molecules were bound per RFC. Order of addition experiments using surface plasmon resonance indicate that RFC forms an ATP-mediated binary complex with PCNA prior to formation of a ternary DNA·PCNA·RFC complex. An ATP-mediated complex between RFC and DNA was not competent for binding PCNA, and the RFC·DNA complex dissociated with hydrolysis of ATP. Based on these experiments a model is proposed in which: (i) RFC binds two ATPs (RFC·ATP2); (ii) this complex binds PCNA (PCNA· RFC·ATP2), which goes through a conformational change to reveal a binding site for one additional ATP (PCNA·RFC·ATP 3); (iii) this complex can bind DNA to yield DNA·PCNA· RFC·ATP3; (iv) a conformational change in the latter complex reveals a fourth binding site for ATP; and (v) the DNA·PCNA· RFC·ATP4 complex is finally competent for completion of PCNA loading and release of RFC upon hydrolysis of ATP.

Original languageEnglish
Pages (from-to)34776-34783
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number37
DOIs
StatePublished - Sep 14 2001

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