Co-operative Binding of Escherichia coli SSB Tetramers to Single-stranded DNA in the (SSB)35 Binding Mode

Ferrari, Marilyn E.; Bujalowski, Wlodzimierz; Lohman, Timothy M.

doi:10.1006/jmbi.1994.1122

Cited by 103 publications

(168 citation statements)

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“…In the case of E. coli SSB, two of the modes differ dramatically in their abilities to bind cooperatively to ssDNA (41,61). In addition, there is a salt-dependent negative cooperativity for ssDNA binding within the E. coli SSB tetramer (67,73,74).…”

Section: Discussionmentioning

confidence: 99%

“…The oligodeoxynucleotides, (dT) L , were synthesized and purified as described (41) and were ≥ 98% pure as judged by denaturing gel electrophoresis. The 5′-Cy3(dT) 29 used for sedimentation studies was further purified by reverse phase HPLC using an Xterra MS C18 Column (Waters, Milford, MA).…”

Section: Scrpa Protein and Nucleic Acidsmentioning

confidence: 99%

“…This view stems primarily from the fact that the first SSB protein that was identified, the phage T4 gene 32 protein, shows highly cooperative binding to ssDNA (1,53). In addition, the E. coli SSB tetramer shows highly cooperative binding in its (SSB) 35 binding mode, although binding is not very cooperative in its fully wrapped (SSB) 65 binding mode (41,61,66). Studies examining the cooperativity of scRPA binding to ssDNA have been reported, although the conclusions have differed significantly.…”

Section: Cooperativity Of Scrpa Binding To M13ssdna Is Lowmentioning

confidence: 99%

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Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

2006

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We have examined the single stranded DNA binding properties of the S. cerevisiae Replication Protein A (scRPA) using fluorescence titrations, isothermal titration calorimetry and sedimentation equilibrium in order to determine whether scRPA can bind to ssDNA in multiple binding modes. We measured the occluded site size for scRPA binding poly(dT), as well as the stoichiometry, equilibrium binding constants and binding enthalpy of scRPA-((dT) L ) complexes as a function of oligodeoxynucleotide length, L. Sedimentation equilibrium studies show that scRPA is stable heterotrimer over the range of [NaCl] examined (0.02 M to 1.5 M). However, the occluded site size, n, undergoes a salt-dependent transition between values of n=18−20 nucleotides at low [NaCl] to n=26 −28 nucleotides at high [NaCl], with a transition midpoint near 0.36 M NaCl (25.0°C, pH 8.1). Measurements of the stoichiometry of scRPA-(dT) L complexes also show a [NaCl]-dependent change in stoichiometry consistent with the observed change in occluded site size. Measurements of the ΔH obs for scRPA binding to (dT) L at 1.5 M NaCl, yield a contact site size of 28 nucleotides, similar to the occluded site size determined at this [NaCl]. Altogether, these data support a model in which scRPA can bind to ssDNA in at least two binding modes, a low site size mode (n = 18 ± 1 nucleotides), stabilized at low [NaCl], in which only three of its OB-folds are used, and a higher site size mode (n = 27 ± 1 nucleotides), stabilized at higher [NaCl], which uses four of its OB-folds. No evidence for highly cooperative binding of scRPA to ssDNA was found either under any conditions examined. Thus, scRPA shows some similar behavior to the E. coli SSB homo-tetramer, which also shows binding mode transitions, but some significant differences also exist.Single stranded (ss) DNA binding (SSB) proteins exist in nearly all organisms and play central roles in all aspects of DNA metabolism, including DNA replication, repair, and recombination. However, the structural forms of SSB proteins differ considerably among different organisms. For example, the bacteriophage T4 gene 32 protein, the first such SSB protein identified (1, 2) is a monomer, whereas the E. coli SSB protein, is a homotetramer (3,4), as are most SSB proteins from eubacteria. In contrast, the eukaryotic SSB protein, commonly called Replication Protein A (RPA) is a hetero-trimer (5,6).In all eukaryotes, RPA consists of three highly conserved subunits of approximately 70, 32, and 14 kDa, named according to their respective molecular weights, and all three subunits are required for function in vivo (5,6). However, RPA homologues display distinct species specificity, such that scRPA from yeast cannot substitute for human RPA (hsRPA) Figure showing the results of agarose gel electrophoresis experiments at low (20 mM) and high (1.5 M) NaCl concentrations for scRPA-ssDNA complexes formed at different protein to DNA ratios indicating low or no cooperativity upon formation of these complexes. This material is availa...

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Section: Discussionmentioning

confidence: 99%

Section: Scrpa Protein and Nucleic Acidsmentioning

confidence: 99%

Section: Cooperativity Of Scrpa Binding To M13ssdna Is Lowmentioning

confidence: 99%

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Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

2006

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“…The NTD consists of an oligosaccharide binding (OB) fold that binds to ssDNA in a sequence independent manner and forms tight inter-domain interactions that stabilise the tetramer in solution. SSB tetramers exhibit a degree of cooperativity upon binding to ssDNA forming extended bead-like structures with the ssDNA wrapped around the beads [2,4] Bacillus subtilis SSBs have been well-characterised and the crystal structure of the NTD of SSB 2 has been solved [5]. Generally, crystallography of bacterial SSB has required either complete or partial removal of the CTD, leading to the assumption that the CTD is intrinsically disordered.…”

Section: Introductionmentioning

confidence: 99%

Defining the Intrinsically Disordered C-Terminal Domain of SSB Reveals DNA-Mediated Compaction

Green

Hatter

Brookes

et al. 2016

Journal of Molecular Biology

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The bacterial single stranded DNA binding protein SSB is a strictly conserved and essential protein involved in diverse functions of DNA metabolism, including replication and repair. SSB comprises a wellcharacterised tetrameric core of N-terminal oligonucleotide binding (OB) folds that bind single-stranded DNA (ssDNA) and four intrinsically disordered C-terminal domains of unknown structure that interact with partner proteins. The generally accepted, albeit speculative, mechanistic model in the field postulates that binding of ssDNA to the OB core induces the flexible, undefined C-terminal arms to expand outwards encouraging functional interactions with partner proteins. In this structural study, we show that the opposite is true. Combined small angle scattering with X-rays and neutrons coupled to coarse-grained modelling reveal that the intrinsically disordered C-terminal arms are relatively collapsed around the tetrameric OB core and collapse further upon ssDNA binding. This implies a mechanism of action, in which the disordered Cterminal domain collapse traps the ssDNA and pulls functional partners onto the ssDNA. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

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“…In vitro, these are salt and protein concentration dependent [6,[12][13][14][15]. The two best characterized of these are the (SSB) 35 mode, in which ~35 nucleotides of ssDNA bind to SSB, interacting with two of the four subunits [16], and the (SSB) 65 mode, in which ~65 nucleotides wrap completely around the tetramer, binding to all four subunits [6,11,15]. The (SSB) 35 mode, which possesses a high degree of positive inter-tetramer cooperativity and forms a continuous coating on DNA [16], tends to be favored at low salt concentrations and high SSB:ssDNA ratios [14,16].…”

Section: Introductionmentioning

confidence: 99%

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Mason

Jergic

et al. 2013

J. Am. Soc. Mass Spectrom.

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Escherichia coli single-stranded DNA-binding protein: nanoESI-MS studies of salt-modulated subunit exchange and DNA binding transactions AbstractSingle-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)70, one molecule of (dT)35, or two molecules of (dT)35] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry. Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNAbinding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT) 70 , one molecule of (dT) 35 or two molecules of (dT) 35 ] was found to prevent SSB subunit exchange.Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

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Co-operative Binding of Escherichia coli SSB Tetramers to Single-stranded DNA in the (SSB)35 Binding Mode

Cited by 103 publications

References 0 publications

Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

Saccharomyces cerevisiae Replication Protein A Binds to Single-Stranded DNA in Multiple Salt-Dependent Modes

Defining the Intrinsically Disordered C-Terminal Domain of SSB Reveals DNA-Mediated Compaction

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

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