title: Structure of the SSB-DNA polymerase III interface and its role in DNA replication.
abstract: Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706-1532, USA.
authors.forename: Aimee H
authors.initials: AH
authors.lastname: Marceau
authors.forename: Soon
authors.initials: S
authors.lastname: Bahng
authors.forename: Shawn C
authors.initials: SC
authors.lastname: Massoni
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: George
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Sandler
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Marians
authors.forename: James L
authors.initials: JL
authors.lastname: Keck
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C103076:DnaX protein, Bacteria; D029968:Escherichia coli Proteins; D020035:Holoenzymes; D004258:DNA Polymerase III