title: Mapping the interaction of DNA with the Escherichia coli DNA polymerase clamp loader complex.
abstract: Sliding clamps are loaded onto DNA by ATP-dependent clamp loader complexes. A recent crystal structure of a clamp loader-clamp complex suggested an unexpected mechanism for DNA recognition, in which the ATPase subunits of the loader spiral around primed DNA. We report the results of fluorescence-based assays that probe the mechanism of the Escherichia coli clamp loader and show that conserved residues clustered within the inner surface of the modeled clamp loader spiral are critical for DNA recognition, DNA-dependent ATPase activity and clamp release. Duplex DNA with a 5'-overhang single-stranded region (corresponding to correctly primed DNA) stimulates clamp release, as does blunt-ended duplex DNA, whereas duplex DNA with a 3' overhang and single-stranded DNA are ineffective. These results provide evidence for the recognition of DNA within an inner chamber formed by the spiral organization of the ATPase domains of the clamp loader.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Goedken
authors.forename: Steven L
authors.initials: SL
authors.lastname: Kazmirski
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Bowman
authors.forename: Mike
authors.initials: M
authors.lastname: O'Donnell
authors.forename: John
authors.initials: J
authors.lastname: Kuriyan
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D021122:Protein Subunits; D004247:DNA; D004259:DNA-Directed DNA Polymerase