Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5' nuclease domain. Removal of the 5' nuclease domains produces the 'large fragment' domains of Pol 1 and Taq, termed Klenow and Klentaq respectively. Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature T(m) of 37 and 100 degrees C respectively at pH 9.5. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5' nuclease domains of each melting separately. The 5' nuclease domain of Taq denatures as a separate peak, 10 degrees C before the Klentaq domain. Melting of the 5' nuclease domain of Pol 1 overlaps with the Klenow fragment. Presence of the 5' nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq. Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins. Melting monitored by CD yields slightly lower T(m) values, but almost identical van't Hoff enthalpy Delta H values, consistent with two-state unfolding followed by an irreversible kinetic step. Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow.

NAD(+)-dependent DNA ligases (LigA) are essential bacterial enzymes that catalyze phosphodiester bond formation during DNA replication and repair processes. Phosphodiester bond formation proceeds through a 3-step reaction mechanism. In the first step, the LigA adenylation domain interacts with NAD(+) to form a covalent enzyme-AMP complex. Although it is well established that the specificity for binding of NAD(+) resides within the adenylation domain, the precise recognition elements for the initial binding event remain unclear. We report here the structure of the adenylation domain from Haemophilus influenzae LigA. This structure is a first snapshot of a LigA-AMP intermediate with NAD(+) bound to domain 1a in its open conformation. The binding affinities of NAD(+) for adenylated and nonadenylated forms of the H. influenzae LigA adenylation domain were similar. The combined crystallographic and NAD(+)-binding data suggest that the initial recognition of NAD(+) is via the NMN binding region in domain 1a of LigA.

e117Pelvic floor disorders and urinary incontinence Poster Presentation Studying the newer TVT-O Abbrevo tape in comparison with the standard TVT-O tape for management of stress urinary incontinenceObjective: To study the newer TVT-O Abbrevo tape for management of SUI in terms of its complications and outcome compared to standard tape.Methods: All the patient who underwent TVT-O abbrevo for Stress Urinary Incontinence in the calendar year 2014 and were followed in the out patient clinic. They were studied for overall success, suboptimal outcome, complete cure and complications. The comparison was made with an Audit done in 2013 for with the standard TVT-O tape with equal number of patient.Place: Midlands Regional Hospital Mullingar Ireland Findings: We found the overall success rate of over 94% with the newer tape with fewer complications, though compared with our own previous audit with the standard tape the success rate was lower but it was not significant. The complete cure rate was as high as 89%. Both these rates were higher than the rates quoted by the tape manufacturing company (Ethicon) in their lone study mentioned on the website.Inference: The newer tape was introduced by Ethicon to reduce post operative complications by decreasing the overall length of the mesh by 38% and rightly so we saw no cases of mesh related complications with comparable outcome with the standard tape. Though the study has its limitations and it was not a controlled trial further studies can be under taken and help us care better for patients by decreasing mesh related complications and its immense implications on the medico-legal costs. http://dx.