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

Roy, Rahul; Козлов, А. Г.; Lohman, Timothy M.; Ha, Taekjip

doi:10.1016/j.jmb.2007.03.079

Cited by 139 publications

(214 citation statements)

References 53 publications

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“…E. coli SSB binds ssDNA in either a highly cooperative mode in which 35 nucleotides are bound per tetramer (SSB 35 ) or a less cooperative mode that binds 65 nucleotides per tetramer (SSB 65 ) (43); these modes can be distinguished in our assay by their differing FRET efficiencies between the fluorescence donor/acceptor pair (Cy3/Cy5) on the DNA substrate (44) (Fig. 3B).…”

Section: Resultsmentioning

confidence: 99%

Structural mechanisms of PriA-mediated DNA replication restart

Bhattacharyya

George

Thurmes

et al. 2013

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

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205

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Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.X-ray crystal structure | single-molecule FRET

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Section: Resultsmentioning

confidence: 99%

Structural mechanisms of PriA-mediated DNA replication restart

Bhattacharyya

George

Thurmes

et al. 2013

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

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205

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“…Given that SSB is known to remain tetrameric in a wide range of solution conditions at concentrations as low as 30 nM [6,53,54], these data reflect the species expected to be present in solution under these conditions. The behavior of 15 N-SSB with regard to ionization efficiency and charge state distribution was indistinguishable from that of unlabeled SSB ( Figures S2a, b).…”

Section: Full-length Ssb Tetramers Undergo Slow But Complete Subunit mentioning

confidence: 95%

“…A shorter total treatment time of 6 h was used, as it was difficult to maintain spectral quality after longer periods in some cases. All three complexes, (SSB/ 15 N-SSB:dT 70 ), (SSB/ 15 N-SSB:dT 35 ), and (SSB/ 15 N-SSB:2dT 35 ) were examined; the 1:1 complexes of SSB with (dT) 70 and (dT) 35 have commonly been used as models for the (SSB) 65 and (SSB) 35 binding modes, respectively [15,30,55,62,63]. In each case, before mixing the SSB and 15 N-SSB complexes, they were analyzed separately by nanoESI-MS to confirm their stoichiometry and the absence of free protein.…”

Section: Binding To Single-stranded Oligonucleotides Abolishes Ssb Sumentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…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%

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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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Sodium Ion‐Induced Structural Transition on the Surface of a DNA‐Interacting Protein

Xu,

Lu,

et al. 2024

Advanced Science

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Protein surfaces have pivotal roles in interactions between proteins and other biological molecules. However, the structural dynamics of protein surfaces have rarely been explored and are poorly understood. Here, the surface of a single‐stranded DNA (ssDNA) binding protein (SSB) with four DNA binding domains that bind ssDNA in binding site sizes of 35, 56, and 65 nucleotides per tetramer is investigated. Using oligonucleotides as probes to sense the charged surface, NaCl induces a two‐state structural transition on the SSB surface even at moderate concentrations. Chelation of sodium ions with charged amino acids alters the network of hydrogen bonds and/or salt bridges on the surface. Such changes are associated with changes in the electrostatic potential landscape and interaction mode. These findings advance the understanding of the molecular mechanism underlying the enigmatic salt‐induced transitions between different DNA binding site sizes of SSBs. This work demonstrates that monovalent salt is a key regulator of biomolecular interactions that not only play roles in non‐specific electrostatic screening effects as usually assumed but also may configure the surface of proteins to contribute to the effective regulation of biomolecular recognition and other downstream events.

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

Cited by 139 publications

References 53 publications

Structural mechanisms of PriA-mediated DNA replication restart

Structural mechanisms of PriA-mediated DNA replication restart

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

Sodium Ion‐Induced Structural Transition on the Surface of a DNA‐Interacting Protein

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