Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

Isolation and visualization of active presynaptic filaments of recA protein and single-stranded DNA

In the presence of ATP, recA protein forms a presynaptic complex with single-stranded DNA that is an obligatory intermediate in homologous pairing. Presynaptic complexes of recA protein and circular single strands that are active in forming joint molecules can be isolated by gel filtration. These isolated active complexes are nucleoprotein filaments with the following characteristics: (i) a contour length that is at least 1.5 times that of the corresponding duplex DNA molecule, (ii) an ordered structure visualized by negative staining as a striated filament with a repeat distance of 9.0 nm and a width of 9.3 nm, (iii) approximately 8 molecules of recA protein and 20 nucleotide residues per striation. The widened spacing between bases in the nucleoprotein filament means that the initial matching of complementary sequences must involve intertwining of the filament and duplex DNA, unwinding of the latter, or some combination of both to equalize the spacing between nascent base pairs. These experiments support the concept that recA protein first forms a filament with single-stranded DNA, which in turn binds to duplex DNA to mediate both homologous pairing and subsequent strand exchange.

Evolution of sequence specificity in a restriction endonuclease by a point mutation

Restriction endonucleases (REases) protect bacteria from invading foreign DNAs and are endowed with exquisite sequence specificity. REases have originated from the ancestral proteins and evolved new sequence specificities by genetic recombination, gene duplication, replication slippage, and transpositional events. They are also speculated to have evolved from nonspecific endonucleases, attaining a high degree of sequence specificity through point mutations. We describe here an example of generation of exquisitely site-specific REase from a highly-promiscuous one by a single point mutation.

Detection,amplification and sequence homology of sodC in clinical isolates of Salmonella sp

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain Salmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonellaenterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X.Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between thesodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028.Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Symmetrical Bisbenzimidazoles with Benzenediyl Spacer: The Role of the Shape of the Ligand on the Stabilization and Structural Alterations in Telomeric G-Quadruplex DNA and Telomerase Inhibition

The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicate senescence. The repeated sequence forming a G-overhang is able to adopt a four-stranded DNA structure called G-quadruplex, which is a poor substrate for the enzyme telomerase. Small molecule based ligands that selectively stabilize the telomeric G-quadruplex DNA, induce telomere shortening eventually leading to cell death. Herein, we have investigated the G-quadruplex DNA interaction with two isomeric bisbenzimidazole-based compounds that differ in terms of shape (V-shaped angular vs linear).While the linear isomer induced some stabilization of the intramolecular G-quadruplex structure generated in the presence of Na+ the other, having V-shaped central planar core, caused a dramatic structural alteration of the latter, above a threshold concentration. This transition was evident from the pronounced changes observed in the circular dichroism spectra and from the get mobility shift assa involving the G-quadruples DNA. Notably, this angular isomer could also induce the G-quadruplex formation in the absence of any added cation. The ligand-quadruples complexes were investigated by computational molecular modeling, providing further information on structure-activity relationships. Finally, TRAP (telomerase repeat amplification protocol) experiments demonstrated that the angular isomer is selective toward the inhibition of telomerase activity.

X-ray and molecular-dynamics studies on Mycobacterium leprae single-stranded DNA-binding protein and comparison with other eubacterial SSB structures

The crystal structures of two forms of Mycobacterium leprae single-stranded DNA-binding protein (SSB) have been determined at 2.05 and 2.8 A resolution. Comparison of these structures with the structures of other eubacterial SSBs indicates considerable variation in their quaternary association, although the DNA-binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but could be related to variation in protein stability. Molecular-dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype Escherichia coli SSB and the individual subunits of both proteins. Together, the X-ray studies and molecular-dynamics simulations yield information on the relatively rigid and flexible regions of the molecule and on the effect of oligomerization on flexibility. The simulations provide insight into the changes in subunit structure on oligomerization. They also provide insight into the stability and time evolution of the hydrogen bonds/water bridges that connect the two pairs of monomers in the tetramer.

A Deterministic Optimization Approach to Protein Sequence Design Using Continuous Models

Determining the sequence of amino acid residues in a heteropolymer chain of a protein with a given conformation is a discrete combinatorial problem that is not generally amenable for gradient-based continuous optimization algorithms. In this paper we present a new approach to this problem using continuous models. In this modeling, continuous "state functions" are proposed to designate the type of each residue in the chain. Such a continuous model helps define a continuous sequence space in which a chosen criterion is optimized to find the most appropriate sequence. Searching a continuous sequence space using a deterministic optimization algorithm makes it possible to find the optimal sequences with much less computation than many other approaches. The computational efficiency of this method is further improved by combining it with a graph spectral method, which explicitly takes into account the topology of the desired conformation and also helps make the combined method more robust. The continuous modeling used here appears to have additional advantages in mimicking the folding pathways and in creating the energy landscapes that help find sequences with high stability and kinetic accessibility. To illustrate the new approach, a widely used simplifying assumption is made by considering only two types of residues: hydrophobic (H) and polar (P). Self-avoiding compact lattice models are used to validate the method with known results in the literature and data that can be practically obtained by exhaustive enumeration on a desktop computer. We also present examples of sequence design for the HP models of some real proteins, which are solved in less than five minutes on a single-processor desktop computer Some open issues and future extensions are noted.

Structural features of B-DNA dodecamer crystal structures: Influence of crystal packing versus base sequence

We have analyzed the set of inter and intra base pair parameters for each dinucleotide step in single crystal structures of dodecamers, solved at high and medium resolution and all crystallized in P2(1)2(1)2(1) space group. The objective was to identify whether all the structures which have either the Drew-Dickerson (DD) sequence d[CGCGAATTCGCG] with some base modification or related sequence (non-DD), would display the same sequence dependent structural variability about its palindromic sequence, despite the molecule being bent at one end because of similar crystal lattice packing effect. Most of the local doublet parameters for base pairs steps G2-C3 and G10-C11 positions, symmetrically situated about the lateral twofold, were significantly correlated between themselves. In non-DD sequences, significant correlations between these positional parameters were absent. The different range of local step parameter values at each sequence position contributed to the gross feature of smooth helix axis bending in all structures. The base pair parameters in some of the positions, for medium resolution DD sequence, were quite unlike the high-resolution set and encompassed a higher range of values. Twist and slide are the two main parameters that show wider conformational range for the middle region of non-DD sequence structures in comparison to DD sequence structures. On the contrary, the minor and major groove features bear good resemblance between DD and non-DD sequence crystal structure datasets. The sugar-phosphate backbone torsion angles are similar in all structures, in sharp contrast to base pair parameter variation for high and low resolution DD and non-DD sequence structures, consisting of unusual (epsilon =g(-), xi =t) B-II conformation at the 10(th) position of the dodecamer sequence. Thus examining DD and non-DD sequence structures packed in the same crystal lattice arrangement, we infer that inter and intra base pair parameters are as symmetrically equivalent in its value as the symmetry related step for the palindromic DD sequence about lateral two-fold axis. This feature would lead us to agree with the conclusion that DNA conformation is not substantially affected by end-to-end or lateral inter-molecular interaction due to crystal lattice packing effect. Non-DD sequence structures acquire step parameter values which reflect the altered sequence at each of the dodecamer sequence position in the orthorhombic lattice while showing similar gross features of DD sequence structures

2(4)-SEMA as a sensitive and offset compensated SLF sequence

Separated Local Field (SLF) spectroscopy is a powerful tool for the determination of structure and dynamics of oriented systems such as membrane proteins oriented in lipid bilayers and liquid crystals. Of many SLF techniques available, Polarization Inversion Spin Exchange at Magic Angle (PISEMA) has found wide application due to its many favorable characteristics. However the pulse sequence suffers from its sensitivity to proton resonance frequency offset. Recently we have proposed a new sequence named 2(4)-SEMA (J. Chem. Phys. 132 (2010) 134301) that overcomes this problem of PISEMA. The present work demonstrates the advantage of 2(4)-SEMA as a highly sensitive SLF technique even for very large proton offset. 2(4)-SEMA has been designed for obtaining reliable dipolar couplings by switching the magic-angle spin-lock for protons over four quadrants as against the use of only two quadrants in PISEMA. It is observed that for on-resonance condition, 2(4)-SEMA gives rise to signal intensity comparable to or slightly higher than that from PISEMA. But under off-resonance conditions, intensities from 2(4)-SEMA are several fold higher than those from PISEMA. Comparison with another offset compensated pulse sequence, SAMPI4, also indicates a better intensity profile for 2(4)-SEMA. Experiments carried out on a single crystal of N-15 labeled N-acetyl-DL-valine and simulations have been used to study the relative performance of the pulse sequences considered. (C) 2010 Elsevier Inc. All rights reserved.

Polypurine-polypyrimidine sequences adopt unwound structure in pBR322 form V DNA as probed by single-hit analysis of HpaII sites.

Structure at the polypurine-polypyrimidine sequences flanking the HpaII sites (CCGG) in pBR322 form V DNA was probed employing single-hit analysis using HpaII restriction endonuclease. Reduced cleavage efficiency of HpaII sites flanked by polypurine-polypyrimidine sequences suggested that under high torsional stress these sequences adopt unwound structures rendering these sites insensitive to restriction enzyme cleavage. In addition to polypurine-polypyrimidine sequences. HpaII sites flanked by alternating purine-pyrimidine sequence, a potential motif of left handed Z-DNA, were also found to be resistant to HpaII cleavage. Results obtained from various studies implicating structure sensitivity of restriction endonucleases and methylases were compiled and a direct correlation was observed between the occurrence of altered sites in a domain and its G/C content in pBR322 form V DNA.

Structure of Sesbania Mosaic Virus at 4·7 Å Resolution and Partial Sequence of the Coat Protein

Sesbania mosaic virus (SMV) is a plant virus infecting Sesbania grandiflora plants in Andhra Pradesh, India. Amino acid sequence of the tryptic peptides of SMV coat protein were determined using a gas phase sequenator. These sequences showed identical amino acids at 69% of the positions when aligned with the corresponding residues of southern bean mosaic virus (SBMV).Crystals diffracting to better than 3 Å resolution were obtained by precipitating the virus with ammonium sulphate. The crystals belonged to rhombohedral space group R3 with α = 291·4 Å and α = 61·9°. Three-dimensional X-ray diffraction data on these crystals were collected to a resolution of 4·7 Å, using a Siemens-Nicolet area detector system. Self-rotation function studies revealed the icosahedral symmetry of the virus particles, as well as their precise orientation in the unit cell. Cross-rotation function and modelling studies with SBMV showed that it is a valid starting model for SMV structure determination. Low resolution phases computed using a polyalanine model of SBMV were subjected to refinement and extension by real-space electron density averaging and solvent flattening. The final electron density map revealed a polypeptide fold similar to SBMV. The single disulphide bridge of SBMV coat protein is retained in SMV. Four icosahedrally independent cation binding sites have been tentatively identified. Three of these sites, related by a quasi threefold axis, are also found in SBMV. The fourth site is situated on the quasi threefold axis. Aspartic acid residues, which replace Ile218 of SBMV from the quasi threefold-related subunits are suitable ligands to the cation at this site

Structure and stability of human telomeric sequence

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)(n). We have investigated the structural properties of the human telomeric repeat oligonucleotide models d(T(2)AG(3))(4), d(G(3)T(2)A)(3)G(3), and d(G(3)T(2)AG(3)) using CD, gel electrophoresis, and chemical probing techniques. The sequences d(G(3)T(2)A)(3)G(3) and d(T(2)AG(3))(4) assume an antiparallel G quartet structure by intramolecular folding, while the sequence d(G(3)T(2)AG(3)) also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide D(G(3)T(2)AG(3)) forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes. Here we show that N-7 of adenine in the hairpin dimer is Hoogsteen hydrogen-bonded. The partial reactivity of loop adenines with DEPC in d(T(2)AG(3))(4) suggests that the intramolecular G quartet structure is highly polymorphic and structures with different loop orientations and topologies are formed in solution. Intra- and interloop hydrogen bonding schemes for the TTA loops are proposed to account for the observed diethyl pyrocarbonate reactivities of adenines. Sodium-induced G quartet structures differ from their potassium-induced counterparts not only in stability but also in loop conformation and interactions. Thus, the overall structure and stability of telomeric sequences are modulated by the cation present, loop sequence, and the number of G tracts, which might be important for the telomere function.

Preferential condensation of SAR-DNA by histone H1 and its SPKK containing octapeptide repeat motif

Linker histone H1 binds preferentially the scaffold associated region (SAR) DNA elements that contain characteristic oligo dA . dT tracts. In the present study, we have compared the condensation brought about by histone H1 of a SAR DNA fragment in the histone spacer region of Drosophila melanogaster with that of a random DNA (pBR322 EcoRI-SalI) fragment by circular dichroism spectroscopy. The condensation of the SAR DNA fragment by histone H1 is 3-4-fold higher than that of the random DNA fragment. A 16-mer peptide, ATPKKSTKKTPKKAKK, the sequence that is present in the C-terminus of histone H1d, which has recently been shown to possess DIVA and chromatin condensing properties, also condenses the SAR DNA fragment preferentially in a highly cooperative manner. We have proposed a model for the dynamics of chromatin structure involving histone H1-SAR DNA interaction through SPKK containing peptide motifs and its competition by AT-hook peptides present in the nonhistone chromosomal proteins like HMG-I and HMG-Y.

Training-Symbol Embedded, High-Rate, Single-Symbol ML-Decodable, Distributed STBCs for Relay Networks

Distributed space-time block codes (DSTBCs) from complex orthogonal designs (CODs) (both square and nonsquare), coordinate interleaved orthogonal designs (CIODs), and Clifford unitary weight designs (CUWDs) are known to lose their single-symbol ML decodable (SSD) property when used in two-hop wireless relay networks using amplify and forward protocol. For such networks, in this paper, three new classes of high rate, training-symbol embedded (TSE) SSD DSTBCs are constructed: TSE-CODs, TSE-CIODs, and TSE-CUWDs. The proposed codes include the training symbols inside the structure of the code which is shown to be the key point to obtain the SSD property along with the channel estimation capability. TSE-CODs are shown to offer full-diversity for arbitrary complex constellations and the constellations for which TSE-CIODs and TSE-CUWDs offer full-diversity are characterized. It is shown that DSTBCs from nonsquare TSE-CODs provide better rates (in symbols per channel use) when compared to the known SSD DSTBCs for relay networks. Important from the practical point of view, the proposed DSTBCs do not contain any zeros in their codewords and as a result, antennas of the relay nodes do not undergo a sequence of switch on/off transitions within every codeword, and, thus, avoid the antenna switching problem.

Chimeras of Escherichia coli and Mycobacterium tuberculosis Single-Stranded DNA Binding Proteins: Characterization and Function in Escherichia coli

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (m beta 1, m beta 1'beta 2, m beta 1-beta 5, m beta 1-beta 6 and m beta 4-beta 5) by transplanting beta 1, beta 1'beta 2, beta 1-beta 5, beta 1-beta 6 and beta 4-beta 5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, m beta 1'beta 2(ESWR) SSB was generated by mutating the MtuSSB specific `PRIY' sequence in the beta 2 strand of m beta 1'beta 2 SSB to EcoSSB specific `ESWR' sequence. Biochemical characterization revealed that except for m beta 1 SSB, all chimeras and a control construct lacking the C-terminal domain (Delta C SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, m beta 1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that m beta 1-beta 6, MtuSSB, m beta 1'beta 2 and m beta 1-beta 5 SSBs complemented E. coli Delta ssb in a dose dependent manner. Complementation by the m beta 1-beta 5 SSB was poor. In contrast, m beta 1'beta 2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.