Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 103, Issue 8, Pages 2546-2551Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0511263103
Keywords
clamp loader; DNA replication
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Funding
- NIGMS NIH HHS [GM32431, GM13306, R01 GM032431, R01 GM013306] Funding Source: Medline
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Numerous proteins that function in DNA metabolic pathways are known to interact with the proliferating cell nuclear antigen (PCNA). The important function of PCNA in stimulating various cellular activities requires its topological linkage with DNA. Loading of the circular PCNA onto duplex DNA requires the activity of a clamp-loader [replication factor C (RFC)] complex and the energy derived from ATP hydrolysis. The mechanistic and structural details regarding PCNA loading by the RFC complex are still developing. In particular, the positive identification of a long-hypothesized structure of an open clamp-RFC complex as an intermediate in loading has remained elusive. In this study, we capture an open yeast PCNA clamp in a complex with RFC through fluorescence energy transfer experiments. We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATP gamma S cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm.
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