The mechanism of Single strand binding protein–RecG binding: Implications for SSB interactome function

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes.     

Sua5p a single‐stranded telomeric DNA‐binding protein facilitates telomere replication

In budding yeast Saccharomyces cerevisiae, telomere length maintenance involves a complicated network as more than 280 telomere maintenance genes have been identified in the nonessential gene deletion mutant set. As a supplement, we identified additional 29 telomere maintenance genes, which were previously taken as essential genes. In this study, we report a novel function of Sua5p in telomere replication. Epistasis analysis and telomere sequencing show that sua5Δ cells display progressively shortened telomeres at early passages, and Sua5 functions downstream telomerase recruitment. Further, biochemical, structural and genetic studies show that Sua5p specifically binds single‐stranded telomeric (ssTG) DNA in vitro through a distinct DNA‐binding region on its surface, and the DNA‐binding ability is essential for its telomere function. Thus, Sua5p represents a novel ssTG DNA‐binding protein and positively regulates the telomere length in vivo.     

RADX interacts with single‐stranded DNA to promote replication fork stability

Single‐stranded DNA (ssDNA) regions form as an intermediate in many DNA‐associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide‐binding (OB) fold domain. The heterotrimeric, multi‐OB fold domain‐containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA‐binding factor in human cells. RADX binds ssDNA via an N‐terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA‐binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA‐binding proteins.     

Surface shapes and surrounding environment analysis of single‐ and double‐stranded DNA‐binding proteins in protein‐DNA interface

Protein‐DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double‐stranded DNA‐binding proteins (DSBs) and single‐stranded DNA‐binding proteins (SSBs) in protein‐DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single‐stranded DNA) or dsDNA (double‐stranded DNA). Proteins 2016; 84:979–989. © 2016 Wiley Periodicals, Inc.     

Identification of centromeric and telomeric DNA‐binding proteins in rice

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G‐rich telomere repeats, respectively. However, the protein components are not fully understood. DNA‐binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA‐specific binding proteins, affinity pull‐down technique was applied in this study to isolate rice centromeric and telomeric DNA‐binding proteins. Fifty‐five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO‐binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA‐binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull‐down technique is effective in the isolation of sequence‐specific binding proteins and will be applicable in future studies of centromere and telomere proteins.     

Ultrafast fluorescence decay profiles reveal differential unstacking of 2-aminopurine from neighboring bases in single-stranded DNA-binding protein subsites

Gene 5 protein (g5p) is a dimeric single-stranded DNA-binding protein encoded by Ff strains of Escherichia coli bacteriophages. The 2-fold rotationally symmetric binding sites of a g5p dimer each bind to four nucleotides, and the dimers bind with high cooperativity to saturate antiparallel single-stranded DNA (ssDNA) strands. Ultrafast time-resolved fluorescence spectroscopies were used to investigate the conformational heterogeneity and dynamics of fluorescent 2-aminopurine (2AP) labels sequestered by bound g5p. The 2AP labels were positioned within the noncomplementary antiparallel tail sequences of d(AC)(8) or d(AC)(9) of hairpin constructs so that each fluorescent label could probe a different subsite location within the DNA-binding site of g5p. Circular dichroism and isothermal calorimetric titrations yielded binding stoichiometries of approximately six dimers per oligomer hairpin when tails were of these lengths. Mobility shift assays demonstrated the formation of a single type of g5p-saturated complex. Femtosecond time-resolved fluorescence spectroscopy showed that the 2AP in the free (non-protein-bound) DNAs had similar heterogeneous distributions of conformations. However, there were significant changes, dominated by a large increase in the population of unstacked bases from ~22 to 59-68%, depending on their subsite locations, when the oligomers were saturated with g5p. Anisotropy data indicated that 2AP in the bound state was less flexible than in the free oligomer. A control oligomer was labeled with 2AP in the loop of the hairpin and showed no significant change in its base stacking upon g5p binding. A proposed model summarizes the data.     

The binding affinity of Ff gene 5 protein depends on the nearest-neighbor composition of the ssDNA substrate

The Ff gene 5 protein (g5p) is considered to be a nonspecific single-stranded DNA binding protein, because it binds cooperatively to and saturates the Ff bacteriophage single-stranded DNA genome and other single-stranded polynucleotides. However, the binding affinity Komega (the intrinsic binding constant times a cooperativity factor) differs by over an order of magnitude for binding to single-stranded polynucleotides such as poly[d(A)] and poly[d(C)]. A polynucleotide that is more stacked, like poly[d(A)], binds more weakly than one that is less stacked, like poly[d(C)]. To test the hypothesis that DNA base stacking, a nearest-neighbor property, is involved in the binding affinity of the Ff g5p for different DNA sequences, Komega values were determined as a function of NaCl concentration for binding to six synthetic sequences 48 nucleotides in length: dA48, dC48, d(AAC)16, d(ACC)16, d(AACC)12, and d(AAACC)9A3. The binding affinities of the protein for these sequences were indeed found to be related to the nearest-neighbor compositions of the sequences, rather than to simple base compositions. That is, the g5p binding site, which is spanned by four nucleotides, discriminates among these sequences on the basis of the relative numbers of nearest neighbors (AA, CC, and AC plus CA) in the sequence. The results support the hypothesis that the extent of base stacking/unstacking of the free, nonbound ssDNA plays an important role in the binding affinity of the Ff gene 5 protein.     

The DNA‐ and RNA‐binding protein FACTOR of DNA METHYLATION 1 requires XH domain‐mediated complex formation for its function in RNA‐directed DNA methylation

Studies have identified a sub‐group of SGS3‐LIKE proteins including FDM1–5 and IDN2 as key components of RNA‐directed DNA methylation pathway (RdDM). Although FDM1 and IDN2 bind RNAs with 5′ overhangs, their functions in the RdDM pathway remain to be examined. Here we show that FDM1 interacts with itself and with IDN2. Gel filtration suggests that FDM1 may exist as a homodimer in a heterotetramer complex in vivo. The XH domain of FDM1 mediates the FDM1–FDM1 and FDM1–IDN2 interactions. Deletion of the XH domain disrupts FDM1 complex formation and results in loss‐of‐function of FDM1. These results demonstrate that XH domain‐mediated complex formation of FDM1 is required for its function in RdDM. In addition, FDM1 binds unmethylated but not methylated DNAs through its coiled‐coil domain. RNAs with 5′ overhangs does not compete with DNA for binding by FDM1, indicating that FDM1 may bind DNA and RNA simultaneously. These results provide insight into how FDM1 functions in RdDM.     

The IDL of E. coli SSB links ssDNA and protein binding by mediating protein–protein interactions

The E. coli single strand DNA binding protein (SSB) is essential to viability where it functions in two seemingly disparate roles: it binds to single stranded DNA (ssDNA) and to target proteins that comprise the SSB interactome. The link between these roles resides in a previously under‐appreciated region of the protein known as the intrinsically disordered linker (IDL). We present a model wherein the IDL is responsible for mediating protein–protein interactions critical to each role. When interactions occur between SSB tetramers, cooperative binding to ssDNA results. When binding occurs between SSB and an interactome partner, storage or loading of that protein onto the DNA takes place. The properties of the IDL that facilitate these interactions include the presence of repeats, a putative polyproline type II helix and, PXXP motifs that may facilitate direct binding to the OB‐fold in a manner similar to that observed for SH3 domain binding of PXXP ligands in eukaryotic systems.     

Potential DNA binding and nuclease functions of ComEC domains characterized in silico

Bacterial competence, which can be natural or induced, allows the uptake of exogenous double stranded DNA (dsDNA) into a competent bacterium. This process is known as transformation. A multiprotein assembly binds and processes the dsDNA to import one strand and degrade another yet the underlying molecular mechanisms are relatively poorly understood. Here distant relationships of domains in Competence protein EC (ComEC) of Bacillus subtilis (Uniprot: P39695) were characterized. DNA‐protein interactions were investigated in silico by analyzing models for structural conservation, surface electrostatics and structure‐based DNA binding propensity; and by data‐driven macromolecular docking of DNA to models. Our findings suggest that the DUF4131 domain contains a cryptic DNA‐binding OB fold domain and that the β‐lactamase‐like domain is the hitherto cryptic competence nuclease. Proteins 2016; 84:1431–1442. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

AtTRB1, a telomeric DNA‐binding protein from Arabidopsis,is concentrated in the nucleolus and shows highly dynamic association with chromatin

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double‐stranded DNA‐binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N‐terminus, a central H1/H5‐like domain and a C‐terminally located coiled‐coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double‐stranded DNA in vitro, while the central H1/H5‐like domain interacts non‐specifically with DNA sequences and mediates protein–protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5‐like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1–GFP localization is cell cycle‐regulated, as the level of nuclear‐associated GFP diminishes during mitotic entry and GFP progressively re‐associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell‐cycle progression, with the other involving rapid exchange.     

Synthesis and DNA‐binding study of imidazole linked thiazolidinone derivatives

A novel series of imidazole‐linked thiazolidinone hybrid molecules were designed and synthesized through a feasible synthetic protocol. The molecules were characterized with Fourier transform infrared (FT‐IR), ¹H nuclear magnetic resonance (NMR), ¹³C NMR and high‐resolution mass spectrometry (HRMS) techniques. In vitro susceptibility tests against Gram‐positive (S. aureus and B. subtilis ) and Gram‐negative bacteria (E. coli and P. aeruginosa ) gave highly promising results. The most active molecule (3e) gave a minimal inhibitory concentration (MIC) value of 3.125 μg/mL which is on par with the reference drug streptomycin. Structure–activity relationships revealed activity enhancement by nitro and chloro groups when they occupied meta position of the arylidene ring in 2‐((3‐(imidazol‐1‐yl)propyl)amino)‐5‐benzylidenethiazolidin‐4‐ones. DNA‐binding study of the most potent molecule 3e with salmon milt DNA (sm‐DNA) under simulated physiological pH was probed with UV–visible absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. These studies established that compound 3e has a strong affinity towards DNA and binds at DNA minor groove with a binding constant (K_b) 0.18 × 10² L mol^?1. Molecular docking simulations predicted strong affinity of 3e towards DNA with a binding affinity (ΔG) ‐8.5 kcal/mol. Van der Waals forces, hydrogen bonding and hydrophobic interactions were predicted as the main forces of interaction. The molecule 3e exhibited specific affinity towards adenine–thiamine base pairs. Copyright © 2016 John Wiley & Sons, Ltd.     

Mannan‐binding lectin reduces CpG DNA‐induced inflammatory cytokine production by human monocytes

Mannan‐binding lectin (MBL) belongs to the collectin family and functions as an opsonin that can also initiate complement activation. Our previous study showed that MBL serves as a double‐stranded RNA binding protein that attenuates polyriboinosinic‐polyribocytidylic acid‐induced TLR3 activation. Prompted by these findings, in the present study cross‐talk between MBL and CpG‐DNA‐induced TLR9 activation was investigated. Here, it was found that MBL also interacts with the TLR9 agonist, CpG oligodeoxynucleotide (CpG‐ODN), in a calcium‐dependent manner. Purified MBL protein suppressed activation of nuclear factor‐kappa B signaling and subsequent production of proinflammatory cytokines from human monocytes induced by CpG‐ODN 2006. These observations indicate that MBL can down‐regulate CpG DNA‐induced TLR9 activation, emphasizing the importance of understanding the interaction of MBL with TLR agonist in host immune defense.     

Statistical analysis of structural determinants for protein–DNA‐binding specificity

DNA‐binding proteins play critical roles in biological processes including gene expression, DNA packaging and DNA repair. They bind to DNA target sequences with different degrees of binding specificity, ranging from highly specific (HS) to nonspecific (NS). Alterations of DNA‐binding specificity, due to either genetic variation or somatic mutations, can lead to various diseases. In this study, a comparative analysis of protein–DNA complex structures was carried out to investigate the structural features that contribute to binding specificity. Protein–DNA complexes were grouped into three general classes based on degrees of binding specificity: HS, multispecific (MS), and NS. Our results show a clear trend of structural features among the three classes, including amino acid binding propensities, simple and complex hydrogen bonds, major/minor groove and base contacts, and DNA shape. We found that aspartate is enriched in HS DNA binding proteins and predominately binds to a cytosine through a single hydrogen bond or two consecutive cytosines through bidentate hydrogen bonds. Aromatic residues, histidine and tyrosine, are highly enriched in the HS and MS groups and may contribute to specific binding through different mechanisms. To further investigate the role of protein flexibility in specific protein–DNA recognition, we analyzed the conformational changes between the bound and unbound states of DNA‐binding proteins and structural variations. The results indicate that HS and MS DNA‐binding domains have larger conformational changes upon DNA‐binding and larger degree of flexibility in both bound and unbound states. Proteins 2016; 84:1147–1161. © 2016 Wiley Periodicals, Inc.     

Diverse modes of 5′‐[4‐(aminoiminomethyl)phenyl]‐[2,2′‐bifuran]‐5‐carboximidamide (DB832) interaction with multi‐stranded DNA structures

The modes of binding of 5′‐[4‐(aminoiminomethyl)phenyl]‐[2,2′‐Bifuran]‐5‐carboximidamide (DB832) to multi‐stranded DNAs: human telomere quadruplex, monomolecular R‐triplex, pyr/pur/pyr triplex consisting of 12 T*(T·A) triplets, and DNA double helical hairpin were studied. The optical adsorption of the ligand was used for monitoring the binding and for determination of the association constants and the numbers of binding sites. CD spectra of DB832 complexes with the oligonucleotides and the data on the energy transfer from DNA bases to the bound DB832 assisted in elucidating the binding modes. The affinity of DB832 to the studied multi‐stranded DNAs was found to be greater (K_ass ≈ 10⁷M^?1) than to the duplex DNA (K_ass ≈ 2 × 10⁵M^?1). A considerable stabilizing effect of DB832 binding on R‐triplex conformation was detected. The nature of the ligand tight binding differed for the studied multi‐stranded DNA depending on their specific conformational features: recombination‐type R‐triplex demonstrated the highest affinity for DB832 groove binding, while pyr/pur/pyr TTA triplex favored DB832 intercalation at the end stacking contacts and the human telomere quadruplex d[AG₃(T₂AG₃)₃] accommodated the ligand in a capping mode. Additionally, the pyr/pur/pyr TTA triplex and d[AG₃(T₂AG₃)₃] quadruplex bound DB832 into their grooves, though with a markedly lesser affinity. DB832 may be useful for discrimination of the multi‐sranded DNA conformations and for R‐triplex stabilization. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 8–20, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Taxonomic distribution,repeats, and functions of the S1 domain‐containing proteins as members of the OB‐fold family

Proteins of the nucleic acid‐binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB‐fold in protein structures. Here, we have analyzed the superfamily of nucleic acid‐binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA‐binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA‐binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain‐containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB‐fold is a distinctive feature of S1 domain‐containing proteins. Proteins from the other families and superfamilies have either one OB‐fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain‐containing proteins. Proteins 2017; 85:602–613. © 2016 Wiley Periodicals, Inc.     

DNA‐binding affinity and nuclease activity of two cytotoxic copper terpyridine complexes

Two copper(II) terpyridine complexes, [Cu(atpy)(NO₃)(H₂O)](NO₃) ? 3H₂O ( 1 ) and [Cu(ttpy)(NO₃)₂] ( 2 ) (atpy = 4′‐p‐N9‐adeninylmethyl‐phenyl‐2,2′:6,2″‐terpyridine; ttpy = 4′‐p‐tolyl‐2,2′:6,2″‐terpyridine) exhibited high cytotoxicity, with average ten times more potency than cisplatin against the human cervix carcinoma cell line (HeLa), the human liver carcinoma cell line (HepG2), the human galactophore carcinoma cell line (MCF7), and the human prostate carcinoma cell line (PC‐3). The cytotoxicity of the complex 1 was lower than that of the complex 2 . Both complexes showed more efficient oxidative DNA cleavage activity under irradiation with UV light at 260 nm than in the presence of ascorbic acid. Especially, complex 1 exhibited evident photoinduced double‐stranded DNA cleavage activity. The preliminary mechanism experiments revealed that hydrogen peroxide was involved in the oxidative DNA damage induced by both complexes. From the absorption titration data, the DNA‐binding affinity of the complexes with surpersoiled plasmid pUC19 DNA, polydAdT, and polydGdC was calculated and complex 2 showed higher binding affinity than complex 1 with all these substrates. The DNA cleavage ability and DNA‐binding affinity of both complexes depended on the substituent group on the terpyrdine ligands. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:295–302, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20292