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J. Biol. Chem., Vol. 277, Issue 49, 47213-47224, December 6, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/49/47213    most recent M206764200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hingorani, M. M. Articles by Coman, M. M. Search for Related Content PubMed PubMed Citation Articles by Hingorani, M. M. Articles by Coman, M. M. Social Bookmarking                      What's this?

On the Specificity of Interaction between the Saccharomyces cerevisiae Clamp Loader Replication Factor C and Primed DNA Templates during DNA Replication^*

Manju M. Hingorani and Maria Magdalena Coman

From the Wesleyan University, Molecular Biology and Biochemistry Department, Middletown, Connecticut 06459

Replication factor C (RFC) catalyzes assembly of circular proliferating cell nuclear antigen clamps around primed DNA, enabling processive synthesis by DNA polymerase during DNA replication and repair. In order to perform this function efficiently, RFC must rapidly recognize primed DNA as the substrate for clamp assembly, particularly during lagging strand synthesis. Earlier reports as well as quantitative DNA binding experiments from this study indicate, however, that RFC interacts with primer-template as well as single- and double-stranded DNA (ssDNA and dsDNA, respectively) with similar high affinity (apparent K_d  10 nM). How then can RFC distinguish primed DNA sites from excess ssDNA and dsDNA at the replication fork? Further analysis reveals that despite its high affinity for various DNA structures, RFC selects primer-template DNA even in the presence of a 50-fold excess of ssDNA and dsDNA. The interaction between ssDNA or dsDNA and RFC is far less stable than between primed DNA and RFC (k_off > 0.2 s¹ versus 0.025 s¹, respectively). We propose that the ability to rapidly bind and release single- and double-stranded DNA coupled with selective, stable binding to primer-template DNA allows RFC to scan DNA efficiently for primed sites where it can pause to initiate clamp assembly.

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