Abstract
In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (polδ), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication. DOI:http://dx.doi.org/10.7554/eLife.00278.001.
Highlights
Replicative DNA polymerases alone are distributive, synthesizing very few nucleotides of complementary DNA before disengaging from an elongating primer strand
A forked DNA substrate in agreement with the minimal requirements for assembly of human replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) onto DNA was labeled with a Cy3 dye that served as the fluorescence resonance energy transfer (FRET) donor (Tsurimoto and Stillman, 1991)
By utilizing FRET experiments to monitor protein-DNA interactions, we were able to monitor the clamp loadingunloading process in real time using recombinant human proteins
Summary
Replicative DNA polymerases (pols) alone are distributive, synthesizing very few nucleotides of complementary DNA before disengaging from an elongating primer strand. Most sliding clamps are closed, requiring an enzyme-catalyzed mechanism which; (1) disrupts an interface within the sliding clamp ring and holds it open for assembly; (2) targets it to primer/template (P/T) junctions where DNA synthesis is initiated; (3) orients it correctly for interaction with pols; and (4) closes it around DNA (Yao et al, 1996; Schurtenberger et al, 1998; Matsumiya et al, 2003; Zhuang et al, 2006; Paschall et al, 2011). Such feats are achieved by heteropentameric complexes referred to as clamp loaders, which utilize ATP to catalyze sitedirected loading of sliding clamps onto DNA
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