Structural insight into the length-dependent binding of ssDNA by SP_0782 from Streptococcus pneumoniae, reveals a divergence in the DNA-binding interface of PC4-like proteins

Abstract SP_0782 from Streptococcus pneumoniae is a dimeric protein that potentially binds with single-stranded DNA (ssDNA) in a manner similar to human PC4, the prototype of PC4-like proteins, which plays roles in transcription and maintenance of genome stability. In a previous NMR study, SP_0782 exhibited an ssDNA-binding property different from YdbC, a prokaryotic PC4-like protein from Lactococcus lactis, but the underlying mechanism remains unclear. Here, we show that although SP_0782 adopts an overall fold similar to those of PC4 and YdbC, the ssDNA length occupied by SP_0782 is shorter than those occupied by PC4 and YdbC. SP_0782 exhibits varied binding patterns for different lengths of ssDNA, and tends to form large complexes with ssDNA in a potential high-density binding manner. The structures of SP_0782 complexed with different ssDNAs reveal that the varied binding patterns are associated with distinct capture of nucleotides in two major DNA-binding regions of SP_0782. Moreover, a comparison of known structures of PC4-like proteins complexed with ssDNA reveals a divergence in the binding interface between prokaryotic and eukaryotic PC4-like proteins. This study provides insights into the ssDNA-binding mechanism of PC4-like proteins, and benefits further study regarding the biological function of SP_0782, probably in DNA protection and natural transformation.

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Rif1 regulates telomere length through conserved HEAT repeats

Nucleic Acids Research ◽

10.1093/nar/gkab206 ◽

2021 ◽

Vol 49 (7) ◽

pp. 3967-3980

Author(s):

Calla B Shubin ◽

Rini Mayangsari ◽

Ariel D Swett ◽

Carol W Greider

Keyword(s):

Dna Binding ◽

Telomere Length ◽

Functional Role ◽

Conserved Residues ◽

Binding Interface ◽

Binding Function ◽

Length Regulation ◽

Telomere Length Regulation ◽

Telomere Regulation ◽

Conserved Amino Acids

AbstractIn budding yeast, Rif1 negatively regulates telomere length, but the mechanism of this regulation has remained elusive. Previous work identified several functional domains of Rif1, but none of these has been shown to mediate telomere length. To define Rif1 domains responsible for telomere regulation, we localized truncations of Rif1 to a single specific telomere and measured telomere length of that telomere compared to bulk telomeres. We found that a domain in the N-terminus containing HEAT repeats, Rif1177–996, was sufficient for length regulation when tethered to the telomere. Charged residues in this region were previously proposed to mediate DNA binding. We found that mutation of these residues disrupted telomere length regulation even when Rif1 was tethered to the telomere. Mutation of other conserved residues in this region, which were not predicted to interact with DNA, also disrupted telomere length maintenance, while mutation of conserved residues distal to this region did not. Our data suggest that conserved amino acids in the region from 436 to 577 play a functional role in telomere length regulation, which is separate from their proposed DNA binding function. We propose that the Rif1 HEAT repeats region represents a protein-protein binding interface that mediates telomere length regulation.

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Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518960113 ◽

2015 ◽

Vol 113 (2) ◽

pp. 326-331 ◽

Cited By ~ 19

Author(s):

William H. Hudson ◽

Bradley R. Kossmann ◽

Ian Mitchelle S. de Vera ◽

Shih-Wei Chuo ◽

Emily R. Weikum ◽

...

Keyword(s):

Transcription Factors ◽

Amino Acid ◽

Dna Binding ◽

Glucocorticoid Receptor ◽

Binding Specificity ◽

Conformational Space ◽

Amino Acid Substitutions ◽

Ancestral Gene ◽

Response Elements ◽

Binding Interface

Many genomes contain families of paralogs—proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD’s ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors’ response element-binding specificity were far from the proteins’ DNA-binding interface and interacted epistatically to change the DBD’s function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.

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Effect of adenine moiety on DNA binding property of copper(ii)–terpyridine complexes

Dalton Transactions ◽

10.1039/b719010g ◽

2008 ◽

pp. 3054 ◽

Cited By ~ 19

Author(s):

Qin Jiang ◽

Zhengyi Wu ◽

Yangmiao Zhang ◽

Anna C. G. Hotze ◽

Michael J. Hannon ◽

...

Keyword(s):

Dna Binding ◽

Binding Property ◽

Dna Binding Property

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Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

10.1101/2021.06.09.447528 ◽

2021 ◽

Author(s):

Bijoya Paul ◽

Loic Chaubet ◽

Emma Verver ◽

Guillermo Montoya

Keyword(s):

Dna Binding ◽

Genome Editing ◽

Optical Tweezers ◽

Target Site ◽

Target Dna ◽

Multiple Binding ◽

Target Sites ◽

Dna Binding And Cleavage ◽

Target Activity ◽

Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

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Metabolic Activation and Covalent Protein Binding of Berberrubine: Insight into the Underlying Mechanism Related to Its Hepatotoxicity

Drug Design Development and Therapy ◽

10.2147/dddt.s274627 ◽

2020 ◽

Vol Volume 14 ◽

pp. 4423-4438

Author(s):

Kai Wang ◽

Jinqiu Rao ◽

Tingting Zhang ◽

Qing Gao ◽

Jichao Zhang ◽

...

Keyword(s):

Protein Binding ◽

Metabolic Activation ◽

Underlying Mechanism ◽

Covalent Protein Binding ◽

Insight Into

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Mechanism of riboregulation of p62 protein oligomerisation by vault RNA1-1 in selective autophagy

10.1101/2021.04.12.439413 ◽

2021 ◽

Author(s):

Magdalena Buescher ◽

Rastislav Horos ◽

Kevin Haubrich ◽

Nikolay Dobrev ◽

Florence Baudin ◽

...

Keyword(s):

Chemical Structure ◽

Membrane Vesicles ◽

Underlying Mechanism ◽

Selective Autophagy ◽

Ubiquitinated Proteins ◽

Flexible Loop ◽

Structure Probing ◽

Molecular Determinants ◽

Necessary And Sufficient ◽

Insight Into

Macroautophagy ensures the clearance of intracellular substrates ranging from single ubiquitinated proteins to large proteotoxic aggregates and defective organelles. The selective autophagy receptor p62 binds these targets and recruits them to double-membrane vesicles, which fuse with lysosomes to degrade their content. We recently uncovered that p62 function is riboregulated by the small non-coding vault RNA1-1. Here, we present detailed insight into the underlying mechanism. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within the RNA that mediates the specific p62 interaction. Our data define molecular determinants that govern mammalian autophagy via the p62-vault RNA1-1 riboregulatory pair.

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A biophysical and structural analysis of DNA binding by oligomeric hSSB1 (NABP2/OBFC2B)

10.1101/2020.08.26.269084 ◽

2020 ◽

Author(s):

Serene El-Kamand ◽

Slobodan Jergic ◽

Teegan Lawson ◽

Ruvini Kariawasam ◽

Derek J. Richard ◽

...

Keyword(s):

Dna Binding ◽

Oxidative Dna Damage ◽

Genomic Stability ◽

Dna Binding Protein ◽

Oxidative Modification ◽

Dna Bases ◽

Genome Integrity ◽

Oligomeric Proteins ◽

Ssdna Binding ◽

Dna Strands

AbstractThe oxidative modification of DNA can result in the loss of genome integrity and must be repaired to maintain overall genomic stability. We have recently demonstrated that human single stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays a crucial role in the removal of 8-oxo-7,8-dihydro- guanine (8-oxoG), the most common form of oxidative DNA damage. The ability of hSSB1 to form disulphide-bonded tetramers and higher oligomers in an oxidative environment is critical for this process. In this study, we have used nuclear magnetic resonance (NMR) spectroscopy and surface plasmon resonance (SPR) experiments to determine the molecular details of ssDNA binding by oligomeric hSSB1. We reveal that hSSB1 oligomers interact with single DNA strands containing damaged DNA bases; however, our data also show that oxidised bases are recognised in the same manner as undamaged DNA bases. We further demonstrate that oxidised hSSB1 interacts with ssDNA with a significantly higher affinity than its monomeric form confirming that oligomeric proteins such as tetramers can bind directly to ssDNA. NMR experiments provide evidence that oligomeric hSSB1 is able to bind longer ssDNA in both binding polarities using a distinct set of residues different to those of the related SSB from Escherichia coli.

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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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