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Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Examination of the ATPase Mechanism of Saccharomyces cerevisiae Replication Factor C

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 heteropentameric protein with four intact ATPase sites. Our analysis of RFC ATP binding indicates that RFC alone binds two 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.

Siying Chen
Manju Hingorani

Molecular Biology & Biochemistry
Wesleyan University
Hall-Atwater Laboratories
Church Street
Middletown, CT 06457, USA

schen01@wesleyan.edu
mhingorani@wesleyan.edu