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

Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.

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Faculty Opinions recommendation of Intrinsically disordered C-terminal tails of E. coli single-stranded DNA binding protein regulate cooperative binding to single-stranded DNA.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725298790.793503146 ◽

2015 ◽

Author(s):

Vladimir Uversky

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Cooperative Binding ◽

Single Stranded Dna ◽

E Coli ◽

Intrinsically Disordered

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Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

Scientific Reports ◽

10.1038/s41598-021-94528-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Krystyna Ślaska-Kiss ◽

Nikolett Zsibrita ◽

Mihály Koncz ◽

Pál Albert ◽

Ákos Csábrádi ◽

...

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Binding Affinity ◽

Dna Methyltransferase ◽

Restriction Enzymes ◽

Dna Binding Protein ◽

E Coli ◽

Dna Binding Affinity ◽

Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

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Antimicrobial Screening, in Silico Studies and QSAR of Chalcone-based 1,4-disubstituted 1,2,3-triazole Hybrids

Drug Research ◽

10.1055/a-1296-7751 ◽

2020 ◽

Author(s):

Pinki Yadav ◽

Kashmiri Lal ◽

Ashwani Kumar

Keyword(s):

Topoisomerase Ii ◽

Antimicrobial Properties ◽

Qsar Model ◽

Quantitative Structure Activity Relationship ◽

Binding Modes ◽

E Coli ◽

Structure Activity ◽

In Silico Studies ◽

Microbial Strains

AbstractThe in vitro antimicrobial properties of some chalcones (1a–1c ) and chalcone tethred 1,4-disubstituted 1,2,3-triazoles (2a–2u) towards different microbial strains viz. Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger and Candida albicans are reported. Compounds 2g and 2u exhibited better potency than the standard Fluconazole with MIC values of 0.0063 µmol/mL and 0.0068 µmol/mL, respectively. Furthermore, molecular docking was performed to investigate the binding modes of two potent compounds 2q and 2g with E. coli topoisomerase II DNA gyrase B and C. albicans lanosterol 14α-demethylase, respectively. Based on these results, a statistically significant quantitative structure activity relationship (QSAR) model was successfully summarized for antibacterial activity against B. subtilis.

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E. coli Single-Stranded DNA Binding (SSB) Protein Undergoes Dynamic Liquid-Liquid Phase Separation Controlled via Protein-Protein and Protein-DNA Interactions

Biophysical Journal ◽

10.1016/j.bpj.2019.11.2680 ◽

2020 ◽

Vol 118 (3) ◽

pp. 484a

Author(s):

Gabor Harami ◽

Zoltan J. Kovacs ◽

János Pálinkás ◽

Rita Pancsa ◽

Veronika Baráth ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Dna Binding ◽

Liquid Phase Separation ◽

Dna Interactions ◽

Single Stranded Dna ◽

E Coli ◽

Protein Dna Interactions ◽

Liquid Liquid Phase Separation

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P VII.20 UV-irradiation induces the expression of a mouse gene encoding a nuclear DNA-binding protein akin to E. coli RecA protein

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis ◽

10.1016/s0027-5107(97)82775-3 ◽

1997 ◽

Vol 379 (1) ◽

pp. S52

Author(s):

P. Kannouche ◽

A. Tissier ◽

G. Pinon-Lataillade ◽

D.S.F. Biard ◽

Ph. Mauffrey ◽

...

Keyword(s):

Dna Binding ◽

Uv Irradiation ◽

Nuclear Dna ◽

Binding Protein ◽

Reca Protein ◽

Dna Binding Protein ◽

Mouse Gene ◽

Gene Encoding ◽

E Coli

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p53 dynamically directs TFIID assembly on target gene promoters

10.1101/083014 ◽

2016 ◽

Cited By ~ 1

Author(s):

R. A. Coleman ◽

Z. Qiao ◽

S. K. Singh ◽

C. S. Peng ◽

M. Cianfrocco ◽

...

Keyword(s):

Dna Binding ◽

Single Molecule ◽

Transcription Initiation ◽

Gene Networks ◽

Target Gene ◽

Core Promoter ◽

Gene Promoters ◽

Binding Modes ◽

Dissociation Kinetics ◽

Target Promoters

AbstractThe p53 tumor suppressor protein is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID’s ability to bind DNA by increasing TFIID contacts with both the core promoter and a region surrounding p53’s response element (RE). Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID’s conversion from a canonical form to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID’s interaction with DNA induces p53 to rapidly dissociate, effectively liberating the RE on the promoter. Collectively, these findings indicate that p53 dynamically escorts and loads the basal transcription machinery onto its target promoters.

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Probing E. coli SSB Protein-DNA topology by reversing DNA backbone polarity

10.1101/2020.12.05.412478 ◽

2020 ◽

Author(s):

Alexander G. Kozlov ◽

Timothy M. Lohman

Keyword(s):

Dna Binding ◽

Crystal Structures ◽

Nearest Neighbor ◽

Genome Maintenance ◽

Binding Properties ◽

Ssdna Binding ◽

E Coli ◽

Phosphodiester Linkage ◽

The Individual ◽

Dna Binding Properties

AbstractE. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homo-tetramers bind ssDNA in two major modes differing in occluded site size and cooperativity. The (SSB)35 mode in which ssDNA wraps on average around two subunits is favored at low [NaCl] and high SSB to DNA ratios and displays high “unlimited”, nearest-neighbor cooperativity forming long protein clusters. The (SSB)65 mode, in which ssDNA wraps completely around four subunits of the tetramer, is favored at higher [NaCl] (> 200 mM) and displays “limited” low cooperativity. Crystal structures of E. coli SSB and P. falciparum SSB show ssDNA bound to the SSB subunits (OB-folds) with opposite polarities of the sugar phosphate backbones. To investigate whether SSB subunits show a polarity preference for binding ssDNA, we examined EcSSB and PfSSB binding to a series of (dT)70 constructs in which the backbone polarity was switched in the middle of the DNA by incorporating a reverse polarity (RP) phosphodiester linkage, either 3’-3’ or 5’-5’. We find only minor effects on the DNA binding properties for these RP constructs, although (dT)70 with a 3’-3’ polarity switch shows decreased affinity for EcSSB in the (SSB)65 mode and lower cooperativity in the (SSB)35 mode. However, (dT)70 in which every phosphodiester linkage is reversed, does not form a completely wrapped (SSB)65 mode, but rather binds EcSSB in the (SSB)35 mode, with little cooperativity. In contrast, PfSSB, which binds ssDNA only in an (SSB)65 mode and with opposite backbone polarity and different topology, shows little effect of backbone polarity on its DNA binding properties. We present structural models suggesting that strict backbone polarity can be maintained for ssDNA binding to the individual OB-folds if there is a change in ssDNA wrapping topology of the RP ssDNA.Statement of SignificanceSingle stranded (ss) DNA binding (SSB) proteins are essential for genome maintenance. Usually homo-tetrameric, bacterial SSBs bind ssDNA in multiple modes, one of which involves wrapping 65 nucleotides of ssDNA around all four subunits. Crystal structures of E. coli and P. falciparum SSB-ssDNA complexes show ssDNA bound with different backbone polarity orientations raising the question of whether these SSBs maintain strict backbone polarity in binding ssDNA. We show that both E. coli and P. falciparum SSBs can still form high affinity fully wrapped complexes with non-natural DNA containing internal reversals of the backbone polarity. These results suggest that both proteins maintain a strict backbone polarity preference, but adopt an alternate ssDNA wrapping topology.

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