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E. coli SSB tetramer binds the first and second molecules of (dT)(35) with heat capacities of opposite sign.

ABSTRACT: We have previously shown that formation of a 1:1 fully wrapped complex of Escherichia coli SSB tetramer with (dT)(70) displays a temperature-dependent sign reversal of the binding heat capacity (?C(P)). Here we examine SSB binding to shorter oligodeoxynucleotides ((dX)(35)) to probe whether this effect requires binding of one or two (dX)(35) molecules per SSB tetramer. We find that the ?C(P) for the first molecule of (dX)(35) is always negative. However, a sign reversal of ?C(P) from negative to positive occurs with increasing temperature for binding of the second (dX)(35). This striking behavior of ?C(P) for the second (dX)(35) appears linked to conformational changes within the ssDNA-SSB complex that are required to form a fully wrapped (SSB)(65) binding mode. These results also underscore that binding heat capacities of macromolecular interactions have multiple origins that cannot be understood simply on the basis of examining static structures.

SUBMITTER: Kozlov AG

PROVIDER: S-EPMC3167024 | biostudies-other | 2011 Nov

REPOSITORIES: biostudies-other

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E. coli SSB tetramer binds the first and second molecules of (dT)(35) with heat capacities of opposite sign.

Kozlov Alexander G AG Lohman Timothy M TM

Biophysical chemistry 20110512 1

We have previously shown that formation of a 1:1 fully wrapped complex of Escherichia coli SSB tetramer with (dT)(70) displays a temperature-dependent sign reversal of the binding heat capacity (ΔC(P)). Here we examine SSB binding to shorter oligodeoxynucleotides ((dX)(35)) to probe whether this effect requires binding of one or two (dX)(35) molecules per SSB tetramer. We find that the ΔC(P) for the first molecule of (dX)(35) is always negative. However, a sign reversal of ΔC(P) from negative to ...[more]

PMID: 21636209

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Project description:Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured the nucleation and growth of RecA--and its eukaryotic homologue RAD51--on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5'?3' direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair, RecOR (RecO and RecR), accelerates both RecA nucleation and filament growth, and that the introduction of RecF further stimulates RecA nucleation.

Dataset Information

E. coli SSB tetramer binds the first and second molecules of (dT)(35) with heat capacities of opposite sign.

Publications

E. coli SSB tetramer binds the first and second molecules of (dT)(35) with heat capacities of opposite sign.

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