Cooperative binding of polyamines induces the Escherichia coli single-strand binding protein-DNA binding mode transitions

Wei, Tai Fen; Bujalowski, Wlodzimierz; Lohman, Timothy M.

doi:10.1021/bi00141a029

Cited by 27 publications

(30 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is worth noting that a spermidine concentration of 50 µM may induce a transition from ( E. coli SSB) 35 to ( E. coli SSB) 65 and thus may prevent the unlimited cooperative binding observation (36). To address this issue, electrophoretic mobility shift assays (EMSA) were performed with increasing E. coli SSB concentration, in spermidine buffer (Tris 20 mM pH 7.5, NaCl 20 mM, SpdCl 3 50 µM) in order to control the gradual formation of M13 ssDNA– E. coli SSB complexes.…”

Section: Resultsmentioning

confidence: 99%

High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein--DNA complexes

Hamon¹,

Pastré²,

Dupaigne³

et al. 2007

Nucleic Acids Research

140

136

View full text Add to dashboard Cite

DNA in living cells is generally processed via the generation and the protection of single-stranded DNA involving the binding of ssDNA-binding proteins (SSBs). The studies of SSB-binding mode transition and cooperativity are therefore critical to many cellular processes like DNA repair and replication. However, only a few atomic force microscopy (AFM) investigations of ssDNA nucleoprotein filaments have been conducted so far. The point is that adsorption of ssDN A–SSB complexes on mica, necessary for AFM imaging, is not an easy task. Here, we addressed this issue by using spermidine as a binding agent. This trivalent cation induces a stronger adsorption on mica than divalent cations, which are commonly used by AFM users but are ineffective in the adsorption of ssDNA–SSB complexes. At low spermidine concentration (<0.3 mM), we obtained AFM images of ssDNA–SSB complexes (E. coli SSB, gp32 and yRPA) on mica at both low and high ionic strengths. In addition, partially or fully saturated nucleoprotein filaments were studied at various monovalent salt concentrations thus allowing the observation of SSB-binding mode transition. In association with conventional biochemical techniques, this work should make it possible to study the dynamics of DNA processes involving DNA–SSB complexes as intermediates by AFM.

show abstract

Section: Resultsmentioning

confidence: 99%

High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein--DNA complexes

Hamon¹,

Pastré²,

Dupaigne³

et al. 2007

Nucleic Acids Research

140

136

View full text Add to dashboard Cite

show abstract

“…The promoting effect exerted by polyamines on the order and rate of formation of the assembly is also indicative. Spermidine was found to stimulate the formation of active RecA-DNA presynaptic filaments by facilitating the removal of the competing singlestranded-binding protein (18). Spermidine was also shown to promote crystallization of purified RecA (19).…”

Section: Discussionmentioning

confidence: 99%

Ordered intracellular RecA–DNA assemblies: A potential site of in vivo RecA-mediated activities

Levin-Zaidman

Frenkiel-Krispin

Shimoni

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. Here we present the first study of the morphological aspects that accompany the SOS response in Escherichia coli. We find that induction of the SOS system in wild-type bacteria results in a fast and massive intracellular coaggregation of RecA and DNA into a lateral macroscopic assembly. The coaggregates comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in vitro RecA-DNA networks, as well as morphological studies of strains carrying RecA mutants, are all consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.biocrystallization ͉ DNA repair ͉ homology search ͉ stress response R ecA-mediated DNA recombination and repair processes proceed through several sequential phases (1-3). A presynaptic filament in which RecA molecules coat a single-stranded DNA substrate is initially formed. The filament acts then as a sequence-specific DNA-binding entity, capable of searching and binding dsDNA sites that are homologous to the RecA-coated segment. Within the resulting joint species, DNA strand exchange and heteroduplex extension processes are promoted. The mechanism that enables a rapid search for DNA homology in vivo, within a highly crowded and complex genome, remains enigmatic.To reach its target, any sequence-specific DNA-binding protein must overcome two general obstacles: a minute cellular concentration of the target, and a vast excess of nontarget, yet still competitive, DNA sites. The search for a homologous DNA site conducted by the RecA-DNA presynaptic filament shares these hurdles, but is further encumbered by the uniquely adverse diffusion characteristics of its components. A DNA target corresponds to a segment that is part of, and embedded within, the chromosome. This, and the large structural asymmetry of DNA, conspire to minimize the diffusion constant of DNA sites (4, 5). In a homology search executed by the RecA-DNA filament, both the searching and the target entities are chromosomal DNA sites whose small diffusion constants drastically attenuate their encounter rate. How then does a RecA-mediated intracellular search evade the kinetic impediments that are intrinsic to the nature of its components?Here we show that damages inflicted on bacterial DNA lead to a rapid formation of an ordered intracellular assembly that accommodates both RecA and DNA. We suggest that the striated morphology of this RecA-DNA assembly is capable of promoting an in vivo homology search by attenuating both the sampling volume and the dimensionality of the process. Moreover, RecA was shown to protect chromosomal DNA from degradation (6) through unknown mechanisms. The tight crystalline packaging that is progressively assumed by the intracellular RecA-DNA assemblies as ...

show abstract

“…In fact, the 35 to 56 transition can also be induced at micromolar concentrations of the polyamine, spermidine. 42 Therefore, charged species present in the cell or a change in the expression level of SSB can easily shift the delicate balance between the two modes, providing the possibility for significant regulatory consequences. We observe that the (SSB) 35 complex can also undergo a conformational transformation to form a previously undetected, low abundance ternary complex (2SSB: DNA), which we term (SSB) 35b .…”

Section: Discussionmentioning

confidence: 99%

Dynamic Structural Rearrangements Between DNA Binding Modes of E. coli SSB Protein

Roy

Козлов

Lohman

et al. 2007

Journal of Molecular Biology

Self Cite

139

193

View full text Add to dashboard Cite

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.

show abstract

Cooperative binding of polyamines induces the Escherichia coli single-strand binding protein-DNA binding mode transitions

Cited by 27 publications

References 40 publications

High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein--DNA complexes

High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein--DNA complexes

Ordered intracellular RecA–DNA assemblies: A potential site of in vivo RecA-mediated activities

Dynamic Structural Rearrangements Between DNA Binding Modes of E. coli SSB Protein

Contact Info

Product

Resources

About