title: On the specificity of interaction between the Saccharomyces cerevisiae clamp loader replication factor C and primed DNA templates during DNA replication.
abstract: 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) approximately 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 s(-1) versus 0.025 s(-1), 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.
authors.affiliation: Wesleyan University, Molecular Biology and Biochemistry Department, Middletown, Connecticut 06459, USA. mhingorani@wesleyan.edu
authors.forename: Manju M
authors.initials: MM
authors.lastname: Hingorani
authors.forename: Maria Magdalena
authors.initials: MM
authors.lastname: Coman
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000096:biosynthesis* / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008954:Models, Biological; D011485:Protein Binding; D051818:Replication Protein C; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004247:DNA; D014357:Trypsin; D051818:Replication Protein C