Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein.

BACKGROUND: Single-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase. RESULTS: We have cloned and purified SSB from Bacillus anthracis (SSB(BA)). In the absence of DNA, at concentrations ≤100 μg/ml, SSB(BA) did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSB(BA) bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)(70) binding suggested that SSB(BA) forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure. CONCLUSIONS: Unlike other prokaryotic SSB proteins, SSB(BA) from Bacillus anthracis appeared to be monomeric at concentrations ≤100 μg/ml as determined by SE-HPLC. SSB(BA) retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSB(BA) appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSB(BA) could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication

Physics Potential of the ICAL detector at the India-based Neutrino Observatory (INO)

The upcoming 50 kt magnetized iron calorimeter (ICAL) detector at the India-based Neutrino Observatory (INO) is designed to study the atmospheric neutrinos and antineutrinos separately over a wide range of energies and path lengths. The primary focus of this experiment is to explore the Earth matter effects by observing the energy and zenith angle dependence of the atmospheric neutrinos in the multi-GeV range. This study will be crucial to address some of the outstanding issues in neutrino oscillation physics, including the fundamental issue of neutrino mass hierarchy. In this document, we present the physics potential of the detector as obtained from realistic detector simulations. We describe the simulation framework, the neutrino interactions in the detector, and the expected response of the detector to particles traversing it. The ICAL detector can determine the energy and direction of the muons to a high precision, and in addition, its sensitivity to multi-GeV hadrons increases its physics reach substantially. Its charge identification capability, and hence its ability to distinguish neutrinos from antineutrinos, makes it an efficient detector for determining the neutrino mass hierarchy. In this report, we outline the analyses carried out for the determination of neutrino mass hierarchy and precision measurements of atmospheric neutrino mixing parameters at ICAL, and give the expected physics reach of the detector with 10 years of runtime. We also explore the potential of ICAL for probing new physics scenarios like CPT violation and the presence of magnetic monopoles.Comment: 139 pages, Physics White Paper of the ICAL (INO) Collaboration, Contents identical with the version published in Pramana - J. Physic

Quantitative analysis of nucleotide modulation of DNA binding by DnaC protein of Escherichia coli.

In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form. DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E. coli DNA replication. In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin. We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC-DNA and DnaC-DnaB interactions using fluorescence anisotropy and affinity sensor analysis. DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides. DnaC bound ssDNA in the complete absence of nucleotides. The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP[S]. In the presence of ADP, the binding affinity decreased approximately fifty-fold. Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch. Both ssDNA and nucleotides modulate DnaB6*DnaC6 complex formation, which has significant implications in DnaC protein function. Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein

Sequence-Dependent Interaction of the Human Papillomavirus E2 Protein with the DNA Elements on Its DNA Replication Origin

The human papillomavirus (HPV) E2 protein is essential for regulating the initiation of viral DNA replication as well as the regulation of transcription of certain HPV-encoded genes. Its ability to recognize and bind to its four recognition sequences in the viral origin is a key step in the initiation of HPV DNA replication. Thus, understanding the mechanism of DNA binding by E2 protein and the unique roles played by individual DNA sequence elements of the replication origin is essential. We have purified the recombinant full-length HPV type 11 E2 protein. Quantitative DNA binding analysis indicated E2 protein bound all four DNA binding sites with reasonably high affinities but with distinct preferences. It bound its cognate binding sites 1, 2, and 4 with higher affinities, but bound binding site 3 with lower affinity. Analysis of binding to these sites unraveled multiple sequence elements that appeared to influence E2 binding affinity and target discrimination, including the sequence of spacer region, flanking sequences, and proximity of E2 binding sites. Thermodynamic analysis indicated hydrophobic interaction in the protein-DNA complex formation. Our studies indicate a large multi-protein complex formation on the HPV-origin DNA, likely due to reasonably high binding affinities as well as intrinsic oligomerization propensity of E2 dimers