Structural plasticity in Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) and its functional implications

17 independent crystal structures of family I uracil-DNA glycosylase from Mycobacterium tuberculosis (MtUng) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 50 position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.

Genetic diversity and proviral DNA load in different neural compartments of HIV-1 subtype C infection

In India, the low prevalence of HIV-associated dementia (HAD) in the Human immunodeficiency virus type 1 (HIV-1) subtype C infection is quite paradoxical given the high-rate of macrophage infiltration into the brain. Whether the direct viral burden in individual brain compartments could be associated with the variability of the neurologic manifestations is controversial. To understand this paradox, we examined the proviral DNA load in nine different brain regions and three different peripheral tissues derived from ten human subjects at autopsy. Using a highly sensitive TaqMan probe-based real-time PCR, we determined the proviral load in multiple samples processed in parallel from each site. Unlike previously published reports, the present analysis identified uniform proviral distribution among the brain compartments examined without preferential accumulation of the DNA in any one of them. The overall viral DNA burden in the brain tissues was very low, approximately 1 viral integration per 1000 cells or less. In a subset of the tissue samples tested, the HIV DNA mostly existed in a free unintegrated form. The V3-V5 envelope sequences, demonstrated a brain-specific compartmentalization in four of the ten subjects and a phylogenetic overlap between the neural and non-neural compartments in three other subjects. The envelope sequences phylogenetically belonged to subtype C and the majority of them were R5 tropic. To the best of our knowledge, the present study represents the first analysis of the proviral burden in subtype C postmortem human brain tissues. Future studies should determine the presence of the viral antigens, the viral transcripts, and the proviral DNA, in parallel, in different brain compartments to shed more light on the significance of the viral burden on neurologic consequences of HIV infection.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising ``multiantigen'' vaccine that elicits robust CMI. IMPORTANCE Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

New dimeric carbazole-benzimidazole mixed ligands for the stabilization of human telomeric G-quadruplex DNA and as telomerase inhibitors. A remarkable influence of the spacer

for selectively targeting cancer cells. Herein, we report the design and evolution of a new kind of carbazole-based benzimidazole dimers for their efficient telomerase inhibition activity. Spectroscopic titrations reveal the ligands high affinity toward the G4 DNA with significantly higher selectivity over duplex-DNA. The electrophoretic mobility shift assay shows that the ligands efficiently promote the formation of 04 DNA even at a lower concentration of the stabilizing K+ ions. The TRAP-LIG assay demonstrates the ligand's potential telomerase inhibition activity and also establishes that the activity proceeds via G4 DNA stabilization. An efficient nuclear internalization of the ligands in several common cancer cells (HeLa, HT1080, and A549) also enabled differentiation between normal HFF cells in co-cultures of cancer and normal ones. The ligands induce significant apoptotic response and antiproliferative activity toward cancer cells selectively when compared to the normal cells.

Selenium-Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway

Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA.

Snaps and mends: DNA breaks and chromosomal translocations

Integrity in entirety is the preferred state of any organism. The temporal and spatial integrity of the genome ensures continued survival of a cell. DNA breakage is the first step towards creation of chromosomal translocations. In this review, we highlight the factors contributing towards the breakage of chromosomal DNA. It has been well-established that the structure and sequence of DNA play a critical role in selective fragility of the genome. Several non-B-DNA structures such as Z-DNA, cruciform DNA, G-quadruplexes, R loops and triplexes have been implicated in generation of genomic fragility leading to translocations. Similarly, specific sequences targeted by proteins such as Recombination Activating Genes and Activation Induced Cytidine Deaminase are involved in translocations. Processes that ensure the integrity of the genome through repair may lead to persistence of breakage and eventually translocations if their actions are anomalous. An insufficient supply of nucleotides and chromatin architecture may also play a critical role. This review focuses on a range of events with the potential to threaten the genomic integrity of a cell, leading to cancer.

DNA Elasticity from Short DNA to Nucleosomal DNA

Active biological processes like transcription, replication, recombination, DNA repair, and DNA packaging encounter bent DNA. Machineries associated with these processes interact with the DNA at short length (<100 base pair) scale. Thus, the study of elasticity of DNA at such length scale is very important. We use fully atomistic molecular dynamics (MD) simulations along with various theoretical methods to determine elastic properties of dsDNA of different lengths and base sequences. We also study DNA elasticity in nucleosome core particle (NCP) both in the presence and the absence of salt. We determine stretch modulus and persistence length of short dsDNA and nucleosomal DNA from contour length distribution and bend angle distribution, respectively. For short dsDNA, we find that stretch modulus increases with ionic strength while persistence length decreases. Calculated values of stretch modulus and persistence length for DNA are in quantitative agreement with available experimental data. The trend is opposite for NCP DNA. We find that the presence of histone core makes the DNA stiffer and thus making the persistence length 3-4 times higher than the bare DNA. Similarly, we also find an increase in the stretch modulus for the NCP DNA. Our study for the first time reports the elastic properties of DNA when it is wrapped around the histone core in NCP. We further show that the WLC model is inadequate to describe DNA elasticity at short length scale. Our results provide a deeper understanding of DNA mechanics and the methods are applicable to most protein-DNA complexes.

Photoinduced DNA Crosslink Formation by Dichloridooxidovanadium(IV) Complexes of Polypyridyl Bases

Oxidovanadium(IV) complexes VO(pyphen)Cl-2] (1) and VO(pydppz)Cl-2] (2), where pyphen is 2-(2-pyridyl)-1,10-phenanthroline and pydppz is 3-(pyridin-2-yl)dipyrido3,2-a:2,3-c]phenazine, show remarkable photoinduced DNA crosslinking ability and photocytotoxicity. The complexes are non-electrolytes in DMF, 1:1 electrolytes in 20% aqueous DMF, and 1:2 electrolytes in 20% aqueous DMF upon photoirradiation with visible light of 400-700 nm. The paramagnetic complexes, which have one unpaired electron, show a d-d band near 780 nm in aqueous DMF. The IR data suggest a V=O moiety trans to a V-N bond. Complex VO(pydppz)Cl-2] (2), as a novel photoinducible nuclear ds-DNA crosslinking agent, shows visible-light-induced cytotoxicity in HeLa and MCF-7 cancer cells by an apoptotic pathway, giving IC50 values of 0.87 +/- 0.07 and 1.4 +/- 0.2 M, respectively, while being essentially nontoxic (IC50 > 40 M) in the dark and less toxic in normal MCF-10A cells.

Mononuclear five-coordinate cobalt(II) complexes with N-4-coordinate pyrazole based ligand and pseudohalogens: synthesis, structures, DNA and protein binding study

A series of mononuclear five-coordinate cobalt(II) complexes, Co(dbdmp)(X)]Y, where dbdmp=N,N-diethyl-N,N-bis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)ethane-1, 2-diamine, X=N-3(-)/NCO-/NCS- and Y=PF6-/BF4-/ClO4-, have been synthesized and characterized by microanalyses and spectroscopic techniques. Crystal structures of Co(N-3)(dbdmp)]PF6 (1), Co(N-3)(dbdmp)]ClO4 (3), Co(NCO)(dbdmp)]PF6 (4), Co(NCO)(dbdmp)]ClO4 (6), and Co(NCS)(dbdmp)]ClO4 (9) have been solved by single-crystal X-ray diffraction studies and showed that all the complexes have distorted trigonal bipyramidal geometry; PF6- counter anion containing complexes Co(N-3)(dbdmp)]PF6 and Co(NCO)(dbdmp)]PF6 have chiral space groups. The binding ability of synthesized complexes with CT-DNA and bovine serum albumin (BSA) has been studied by spectroscopic methods and viscosity measurements. The experimental results of absorption titration of cobalt(II) complexes with CT-DNA indicate that the complexes have ability to form adducts and they can stabilize the DNA helix. The cobalt(II) complexes exhibit good binding propensity to BSA protein.

A novel benzimidazole derivative binds to the DNA minor groove and induces apoptosis in leukemic cells

DNA minor groove binders are an important class of chemotherapeutic agents. These small molecule inhibitors interfere with various cellular processes like DNA replication and transcription. Several benzimidazole derivatives showed affinity towards the DNA minor groove. In this study we show the synthesis and biological studies of a novel benzimidazole derivative (MH1), that inhibits topoisomerase II activity and in vitro transcription. UV-visible and fluorescence spectroscopic methods in conjunction with Hoechst displacement assay demonstrate that MH1 binds to DNA at the minor groove. Cytotoxic studies showed that leukemic cells are more sensitive to MH1 compared to cancer cells of epithelial origin. Further, we find that MH1 treatment leads to cell cycle arrest at G2/M, at early time points in Molt4 cells. Finally multiple cellular assays demonstrate that MH1 treatment leads to reduction in MMP, induction of apoptosis by activating CASPASE 9 and CASPASE 3. Thus our study shows MH1, a novel DNA minor groove binder, induces cytotoxicity efficiently in leukemic cells by activating the intrinsic pathway of apoptosis.

Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart

Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis.

A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes.