Structural mechanisms of SLF1 interactions with Histone H4 and RAD18 at the stalled replication fork.

DNA damage that obstructs the replication machinery poses a significant threat to genome stability. Replication-coupled repair mechanisms safeguard stalled replication forks by coordinating proteins involved in the DNA damage response (DDR) and replication. SLF1 (SMC5-SMC6 complex localization factor 1) is crucial for facilitating the recruitment of the SMC5/6 complex to damage sites through interactions with SLF2, RAD18, and nucleosomes.

Enhancing research culture through PhD training: a systems approach to identifying leverage points for policy formation.

This article examines the role of PhD training programmes in identifying and implementing positive interventions in research culture in the biosciences. Using a data set consisting of transcripts from interviews and group discussions with 179 participants from 18 of the current 23 (78%) UK-based Wellcome-funded PhD programmes, we apply a systems theory methodology to the system of higher education and PhD training. Using system mapping as an investigative tool, this approach identifies points of leverage within the system where policy interventions might be best targeted to affect changes to research culture in the global higher education sector.

Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine.

Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified.

Mechanistic insights into the enzymatic activity and inhibition of the replicative polymerase exonuclease domain from Mycobacterium tuberculosis.

DNA replication fidelity maintains low mutation rates in bacteria. The ε-subunit of a replisome generally acts as the main proofreader during replication, using its 3'-5' exonuclease activity to excise misincorporated bases thereby maintaining faithful replication. In Mycobacterium tuberculosis (Mtb), however, the polymerase and histidinol phosphatase (PHP) domain of the DNA polymerase DnaE1 is the primary proofreader.

Crystal structures of Mycobacterium tuberculosis HspAT and ArAT reveal structural basis of their distinct substrate specificities.

Aminotransferases of subfamily Iβ, which include histidinol phosphate aminotransferases (HspATs) and aromatic amino acid aminotransferases (ArATs), are structurally similar but possess distinct substrate specificities. This study, encompassing structural and biochemical characterisation of HspAT and ArAT from Mycobacterium tuberculosis demonstrates that the residues lining the substrate binding pocket and N-terminal lid are the primary determinants of their substrate specificities. In mHspAT, hydrophilic residues in the substrate binding pocket and N-terminal lid allow the entry and binding of its preferential substrate, Hsp.

Structures of native, substrate-bound and inhibited forms of Mycobacterium tuberculosis imidazoleglycerol-phosphate dehydratase.

Imidazoleglycerol-phosphate dehydratase (IGPD; HisB), which catalyses the conversion of imidazoleglycerol-phosphate (IGP) to imidazoleacetol-phosphate in the histidine biosynthesis pathway, is absent in mammals. This feature makes it an attractive target for herbicide discovery. Here, the crystal structure of Mycobacterium tuberculosis (Mtb) IGPD is reported together with the first crystal structures of substrate-bound and inhibited (by 3-amino-1,2,4-triazole; ATZ) forms of IGPD from any organism.

Sample preparation, crystallization and structure solution of HisC from Mycobacterium tuberculosis.

Histidinolphosphate aminotransferase (HisC; Rv1600) from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals suitable for X-ray analysis were grown by the hanging-drop vapour-diffusion technique using 30% polyethylene glycol monomethyl ether 2000 as the precipitant.

Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction studies of histidinol phosphate aminotransferase (HisC2) from Mycobacterium tuberculosis.

HisC2 from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion technique from a condition consisting of 7 mg ml(-1) HisC2 (in 20 mM Tris pH 8.

HisB from Mycobacterium tuberculosis: cloning, overexpression in Mycobacterium smegmatis, purification, crystallization and preliminary X-ray crystallographic analysis.

HisB, encoded by open reading frame Rv1601, possesses enzymatic activity as an imidazoleglycerol-phosphate dehydratase in the histidine-biosynthetic pathway of Mycobacterium tuberculosis. A recombinant form of HisB was crystallized in three crystal forms: crystals grown using 20% PEG 1500 as a precipitant belonged to either the cubic space group P432 or the tetragonal space group P4, while an orthorhombic crystal form belonging to space group P2(1)2(1)2 was obtained using 15% PEG 5000 and 10 mM MnCl(2) as precipitant. The structure of HisB in the orthorhombic crystal form was solved by the molecular-replacement method using the crystal structure of its Arabidopsis thaliana counterpart, which shares 47% sequence identity with Rv1601, as the search model.