Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Antony, Edwin; Weiland, Elizabeth; Yuan, Quan; Manhart, Carol M.; Nguyen, Binh; Козлов, А. Г.; McHenry, Charles S.; Lohman, Timothy M.

doi:10.1016/j.jmb.2013.08.021

Cited by 66 publications

(73 citation statements)

References 89 publications

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“…Since removal of the CTD moved the peak, this confirms that it is exclusively the CTDs (residues 107-171) that contribute to this flexibility in solution. This result is in line with previous observations that the SSB CTD has flexible properties (as predicted through difficulties with protein crystallisation and secondary structure predictions) and the observation that the NTD forms a rigid tetramer with no major flexibility [3,6]. Addition of dT35 or dT70 at one-fold molar excess over tetramer does not change the shape of the curve suggesting that despite compaction, the CTD remains somewhat mobile.…”

Section: Distance Distribution Functions -P(r) Plots -(Figsupporting

confidence: 91%

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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Section: Distance Distribution Functions -P(r) Plots -(Figsupporting

confidence: 91%

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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“…We also found that DnaN levels were increased, suggesting that dnaN is damage inducible. We suggest that the basal recOR-independent RecA localization we observe in ssb⌬35 cells is caused by elevated RecA levels in vivo, which may be caused or exacerbated by a decrease in the ability of ssb⌬35 to bind ssDNA (62).…”

Section: Resultsmentioning

confidence: 79%

RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

et al. 2014

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bRecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify singlestranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.

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“…The EcoSSB contains a nine-amino acid sequence (C-terminal tail) that binds to more than a dozen different proteins that it recruits to their sites of function in DNA replication, repair, and recombination (1,21), and it regulates the ssDNA binding affinity and cooperativity between binding sites (22). In human mtSSB, this C-terminal tail is completely missing (5), which raises the questions of whether mtSSB may show distinct ssDNA binding modes and whether the kinetic and thermodynamic basis for formation of protein-DNA complexes is different from that for EcoSSB.…”

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confidence: 99%

The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange

Qian

Johnson

2017

Journal of Biological Chemistry

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The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB-ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB) 30 and (SSB) 60 , defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB) 60 and (SSB) 30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 ؋ 10 9 M ؊1 s ؊1 ) and that longer DNA (>60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein.

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Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Cited by 66 publications

References 89 publications

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

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

RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange

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