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Examination of the ATPase Mechanism of Saccharomyces cerevisiae Replication Factor C
Author(s) -
Chen Siying,
Hingorani Manju
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a658-d
Subject(s) - dna clamp , atp hydrolysis , primer (cosmetics) , dna polymerase , dna replication , replication factor c , dna , biophysics , biochemistry , processivity , atpase , proliferating cell nuclear antigen , chemistry , biology , microbiology and biotechnology , enzyme , eukaryotic dna replication , reverse transcriptase , gene , rna , organic chemistry
Processive replication is achieved by tethering the polymerase to the DNA template by a circular sliding clamp protein. Clamps are loaded onto primer‐template DNA by clamp loaders in a reaction fueled by ATP hydrolysis. Clamp loaders are pentameric AAA+ protein complexes conserved across all domains of life. Replication Factor C, the S. cerevisiae clamp loader, is a hetero‐pentameric protein with 4 intact ATPase sites. Our analysis of RFC ATP binding indicates that RFC alone binds 2 ATP molecules and in the presence of PCNA clamp and/or primer‐template DNA, it binds 3–4 ATP molecules, which indicates a link between the ATP binding and PCNA/DNA binding activities. According to results from our pre‐steady state kinetic analysis, RFC alone hydrolyzes ATP at a limiting rate of 0.025 second −1 , which increases to 0.05 second −1 in the presence of PCNA clamp. PCNA stimulates formation of an “active” RFC‐ATP‐PCNA complex within 2 seconds while it takes 5–7 second for an RFC‐ATP binary complex alone to achieve the same “active” conformation. The binding of double‐stranded DNA to this complex, as well as primer‐template DNA with either recessed 3′ or 5′ends, triggers rapid hydrolysis of 3 ATP molecules at a rate of 7–10 second −1 while binding of single‐stranded DNA does not trigger rapid ATP hydrolysis. This suggests that RFC “recognizes” the duplex portion of primer‐template DNA for clamp assembly.