Cooperative kinking at distant sites in mechanically stressed DNA.

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.

Indicators of therapeutic effect in FIT-06, a Phase II trial of a DNA vaccine, GTU(®)-Multi-HIVB, in untreated HIV-1 infected subjects.

BACKGROUND: Combination highly active antiretroviral therapy (HAART) has significantly decreased HIV-1 related morbidity and mortality globally transforming HIV into a controllable condition. HAART has a number of limitations though, including limited access in resource constrained countries, which have driven the search for simpler, affordable HIV-1 treatment modalities. Therapeutic HIV-1 vaccines aim to provide immunological support to slow disease progression and decrease transmission. We evaluated the safety, immunogenicity and clinical effect of a novel recombinant plasmid DNA therapeutic HIV-1 vaccine, GTU(®)-multi-HIVB, containing 6 different genes derived from an HIV-1 subtype B isolate. METHODS: 63 untreated, healthy, HIV-1 infected, adults between 18 and 40 years were enrolled in a single-blinded, placebo-controlled Phase II trial in South Africa. Subjects were HIV-1 subtype C infected, had never received antiretrovirals, with CD4 ≥ 350 cells/mm(3) and pHIV-RNA ≥ 50 copies/mL at screening. Subjects were allocated to vaccine or placebo groups in a 2:1 ratio either administered intradermally (ID) (0.5mg/dose) or intramuscularly (IM) (1mg/dose) at 0, 4 and 12 weeks boosted at 76 and 80 weeks with 1mg/dose (ID) and 2mg/dose (IM), respectively. Safety was assessed by adverse event monitoring and immunogenicity by HIV-1-specific CD4+ and CD8+ T-cells using intracellular cytokine staining (ICS), pHIV-RNA and CD4 counts. RESULTS: Vaccine was safe and well tolerated with no vaccine related serious adverse events. Significant declines in log pHIV-RNA (p=0.012) and increases in CD4+ T cell counts (p=0.066) were observed in the vaccine group compared to placebo, more pronounced after IM administration and in some HLA haplotypes (B*5703) maintained for 17 months after the final immunisation. CONCLUSIONS: The GTU(®)-multi-HIVB plasmid recombinant DNA therapeutic HIV-1 vaccine is safe, well tolerated and favourably affects pHIV-RNA and CD4 counts in untreated HIV-1 infected individuals after IM administration in subjects with HLA B*57, B*8101 and B*5801 haplotypes.

Differential involvement of MEK kinase 1 (MEKK1) in the induction of apoptosis in response to microtubule-targeted drugs versus DNA damaging agents.

MEK kinase 1 (MEKK1) is a 196-kDa enzyme that is involved in the regulation of the c-Jun N-terminal kinase (JNK) pathway and apoptosis. In cells exposed to genotoxic agents including etoposide and cytosine arabinoside, MEKK1 is cleaved at Asp874 by caspases. The cleaved kinase domain of MEKK1, itself, stimulates caspase activity leading to apoptosis. Kinase-inactive MEKK1 expressed in HEK293 cells effectively blocks genotoxin-induced apoptosis. Treatment of cells with taxol, a microtubule stabilizing agent, did not induce MEKK1 cleavage in cells, and kinase-inactive MEKK1 expression failed to block taxol-induced apoptosis. MEKK1 became activated in HEK293 cells exposed to taxol, but in contrast to etoposide-treatment, taxol failed to increase JNK activity. Taxol treatment of cells, therefore, dissociates MEKK1 activation from the regulation of the JNK pathway. Overexpression of anti-apoptotic Bcl2 blocked MEKK1 and taxol-induced apoptosis but did not block the caspase-dependent cleavage of MEKK1 in response to etoposide. This indicates Bcl2 inhibition of apoptosis is, therefore, downstream of caspase-dependent MEKK1 cleavage. The results define the involvement of MEKK1 in the induction of apoptosis by genotoxins but not microtubule altering drugs.