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Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein.

ABSTRACT: Escherichia coli single-stranded (ss)DNA binding (SSB) protein binds ssDNA in multiple binding modes and regulates many DNA processes via protein-protein interactions. Here, we present direct evidence for fluctuations between the two major modes of SSB binding, (SSB)(35) and (SSB)(65) formed on (dT)(70), with rates of interconversion on time scales that vary as much as 200-fold for a mere fourfold change in NaCl concentration. Such remarkable electrostatic effects allow only one of the two modes to be significantly populated outside a narrow range of salt concentration, providing a context for precise control of SSB function in cellular processes via SSB expression levels and interactions with other proteins. Deletion of the acidic C terminus of SSB, the site of binding of several proteins involved in DNA metabolism, does not affect the strong salt dependence, but shifts the equilibrium towards the highly cooperative (SSB)(35) mode, suggesting that interactions of proteins with the C terminus may regulate the binding mode transition and vice versa. Single molecule analysis further revealed a novel low abundance binding configuration and provides a direct demonstration that the SSB-ssDNA complex is a finely tuned assembly in dynamic equilibrium among several well-defined structural and functional states.

SUBMITTER: Roy R

PROVIDER: S-EPMC2041828 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein.

Roy Rahul R Kozlov Alexander G AG Lohman Timothy M TM Ha Taekjip T

Journal of molecular biology 20070405 5

Escherichia coli single-stranded (ss)DNA binding (SSB) protein binds ssDNA in multiple binding modes and regulates many DNA processes via protein-protein interactions. Here, we present direct evidence for fluctuations between the two major modes of SSB binding, (SSB)(35) and (SSB)(65) formed on (dT)(70), with rates of interconversion on time scales that vary as much as 200-fold for a mere fourfold change in NaCl concentration. Such remarkable electrostatic effects allow only one of the two modes ...[more]

PMID: 17490681

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Project description:Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes differing in occluded site size and cooperativity. The 35-site-size ((SSB)35) mode favored at low [NaCl] and high SSB/DNA ratios displays high "unlimited" nearest-neighbor cooperativity (?35), forming long protein clusters, whereas the 65-site-size ((SSB)65) mode in which ssDNA wraps completely around the tetramer is favored at higher [NaCl] (>200 mM) and displays "limited" cooperativity (?65), forming only dimers of tetramers. In addition, a non-nearest-neighbor high cooperativity can also occur in the (SSB)65 mode on long ssDNA even at physiological salt concentrations in the presence of glutamate and requires its intrinsically disordered C-terminal linker (IDL) region. However, whether cooperativity exists between the different modes and the role of the IDL in nearest-neighbor cooperativity has not been probed. Here, we combine sedimentation velocity and fluorescence titration studies to examine nearest-neighbor cooperativity in each binding mode and between binding modes using (dT)70 and (dT)140. We find that the (SSB)35 mode always shows extremely high "unlimited" cooperativity that requires the IDL. At high salt, wild-type SSB and a variant without the IDL, SSB-?L, bind in the (SSB)65 mode but show little cooperativity, although cooperativity increases at lower [NaCl] for wild-type SSB. We also find significant intermode nearest-neighbor cooperativity (?65/35), with ?65 ? ?65/35 <?35. The intrinsically disordered region of SSB is required for all cooperative interactions; however, in contrast to the non-nearest-neighbor cooperativity observed on longer ssDNA, glutamate does not enhance these nearest-neighbor cooperativities. Therefore, we show that SSB possesses four types of cooperative interactions, with clear differences in the forces stabilizing nearest-neighbor versus non-nearest-neighbor cooperativity.

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Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein.

Publications

Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein.

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