Characterization of Agrobacterium tumefaciens DNA ligases C and D

Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3′-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3′-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3′-OH end. The PE domains also have a 3′-phosphatase activity on an all-DNA primer-template that yields a 3′-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ

An adaptive grid air model for urban to regional scale air quality problems

Issued as final reportUnited States. Environmental Protection Agenc

Structure-guided Mutational Analysis of the OB, HhH, and BRCT Domains of Escherichia coli DNA Ligase*S⃞

NAD+-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg333); penetrate the minor grove and distort the nick (Val383 and Ile384); or stabilize the OB fold (Arg379). The essential constituents of the HhH domain include: four glycines (Gly455, Gly489, Gly521, Gly553), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg487, which penetrates the minor groove at the outer margin on the 3 ®-OH side of the nick; and Arg446, which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation

Esterification of free fatty acids in used cooking oil using ion-exchange resins as catalysts: An efficient pretreatment method for biodiesel feedstock

The esterification of used cooking oil (UCO) with methanol was studied using different types of ion-exchange resins, that is, Purolite D5081, Purolite D5082, and Amberlyst 36. Several catalyst characterization analyses (elemental analysis, surface area measurement, particle size distribution analysis, scanning electron microscopy analysis, true density measurement, and acid capacity analysis) have been conducted in the screening stage. Of all of the catalysts investigated, Purolite D5081 resin showed the best catalytic performance and was selected for further experimental studies. The esterification process was carried out in a jacketed stirred batch reactor for 8 h. Elimination of mass transfer resistances and the effect of catalyst loading (0.5–1.5% w/w), reaction temperature (50–65 °C), and methanol to UCO feed mole ratio (4:1–12:1) on the conversion of FFAs were investigated. The highest FFAs conversion was found to be 92%, at a catalyst loading of 1.25% w/w, 60 °C reaction temperature, 6:1 methanol to UCO molar ratio, and stirring speed of 475 rpm. During the reusability study, the conversion of catalyst dropped by 8–10% after each reutilization cycle. Several experiments have been conducted through the homogeneous contribution study, and the results confirmed that both resin pore blockage and sulfur leaching are dominant factors that decrease the catalytic performance of Purolite D5081 ion-exchange resin