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DNA polymerase III, a decameric 420-kDa assembly, simultaneously replicates both strands of the chromosome in Escherichia coli. A subassembly of this holoenzyme, the seven-subunit clamp loader complex, is responsible for loading the sliding clamp (β2) onto DNA. Here, we use structural information derived from ion mobility mass spectrometry (IM-MS) to build three-dimensional models of one form of the full clamp loader complex, γ3δδ'ψχ (254 kDa). By probing the interaction between the clamp loader and a single-stranded DNA (ssDNA) binding protein (SSB4) and by identifying two distinct conformational states, with and without ssDNA, we assemble models of ψχ-SSB4 (108 kDa) and the clamp loader-SSB4 (340 kDa) consistent with IM data. A significant increase in measured collision cross-section (~10%) of the clamp loader-SSB4 complex upon DNA binding suggests large conformational rearrangements. This DNA bound conformation represents the active state and, along with the presence of ψχ, stabilises the clamp loader-SSB4 complex. Overall, this study of a large heteromeric complex analysed by IM-MS, coupled with integrative modelling, highlights the potential of such an approach to reveal structural features of previously unknown complexes of high biological importance.

Original publication

DOI

10.1016/j.jmb.2013.04.006

Type

Journal article

Journal

J Mol Biol

Publication Date

29/11/2013

Volume

425

Pages

4790 - 4801

Keywords

ATD, CCS, CG, DNA replication, IM, MS, PA, PDB, Protein Data Bank, USR, arrival time distribution, clamp loader, coarse grained, collision cross-section, integrative modelling, ion mobility, ion mobility-mass spectrometry, mass spectrometry, projection approximation, single-stranded DNA, single-stranded DNA binding protein (SSB), ssDNA, ultrafast shape recognition, DNA Polymerase III, DNA, Single-Stranded, DNA-Binding Proteins, Escherichia coli, Imaging, Three-Dimensional, Mass Spectrometry, Models, Molecular, Multienzyme Complexes