Summary The nonrandom distribution of meiotic recombination shapes patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in accumulation of Spo11 protein covalently bound to small DNA fragments. We show here that sequencing these fragments provides a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore the influence of large-scale chromosome structures, chromatin, transcription factors, and local sequence composition on DSB distributions. Our analysis supports the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. Mechanistic aspects of DSB formation and early processing steps are also uncovered. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We discuss implications for evolutionary dynamics of recombination hotspots.

The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins.

We report the 2.4 Å crystal structure for lipoamide dehydrogenase encoded by lpdC from Mycobacterium tuberculosis. Based on the Lpd structure and sequence alignment between bacterial and eukaryotic Lpd sequences, we generated single point mutations in Lpd and assayed the resulting proteins for their ability to catalyze lipoamide reduction/oxidation alone and in complex with other proteins that participate in pyruvate dehydrogenase and peroxidase activities. The results suggest that amino acid residues conserved in mycobacterial species but not conserved in eukaryotic Lpd family members modulate either or both activities and include Arg-93, His-98, Lys-103, and His-386. In addition, Arg-93 and His-386 are involved in forming both "open" and "closed" active site conformations, suggesting that these residues play a role in dynamically regulating Lpd function. Taken together, these data suggest protein surfaces that should be considered while developing strategies for inhibiting this enzyme.Over the last decade, tuberculosis has reemerged as one of the leading causes of death (1). Despite the substantive response of the host immune system that includes both oxidative and nitrosative stress, Mycobacterium tuberculosis (Mtb) 2 evades these insults, in part by adopting metabolic enzymes to catabolize these toxic compounds (2). Lipoamide dehydrogenase (Lpd), dihydrolipoamide acyltransferase (DlaT; formerly termed succinyl transferase), an alkylhydroperoxidase termed AhpC, and the protein (AhpD) encoded by an adjacent gene, have been shown to take part in this defense pathway (3, 4). All of these enzymes are oxidoreductases, and each contains redox centers that reduce or oxidize adjacent partners in the pathway.The Lpd redox center consists of two conserved cysteine residues and a non-covalently bound FAD molecule (5). DlaT, encoded by the gene Rv2215, was originally annotated as the E2 component of ␣-ketoglutarate dehydrogenase, but subsequent investigation revealed that Mtb lacks ␣-ketoglutarate dehydrogenase. Rv2215 instead encodes DlaT, the E2 component of pyruvate dehydrogenase (PDH) (6). DlaT contains a redox center in the form of dihydrolipoyl cofactors that are covalently attached to lysine ⑀-amino groups within lipoyl domains that form the so-called "swinging arms" of this enzyme (7). Both AhpD and AhpC contain redox centers consisting of two conserved cysteine residues (3). Each of these redox centers can oxidize or reduce the redox center of the adjacent enzyme in the cascade. For example, AhpC metabolizes peroxides and peroxynitrite (ROI/RNI) (3, 8, 9), AhpD regenerates the redox center of AhpC, DlaT regenerates the redox center of AhpD, and Lpd regenerates the redox center of DlaT. Lpd serves a critical function in this cascade because its redox center is regenerated directly by NADH. The process can be represented as a flow of electrons from NADH 3 Lpd 3 DlaT 3 AhpD 3 AhpC 3 ROOH. The absence or perturbation of any member in this cascade has been shown to negatively impact the electron flow (3), 3 thus ma...

BackgroundThere is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.ResultsHere we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration.ConclusionsWe present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.Electronic supplementary materialThe online version of this article (10.1186/s12896-017-0393-y) contains supplementary material, which is available to authorized users.

Mek1 is a CHK2/Rad53-family kinase that regulates meiotic recombination and progression upon its activation in response to DNA double-strand breaks (DSBs). The full catalog of direct Mek1 phosphorylation targets remains unknown. Here, we show that phosphorylation of histone H3 on threonine 11 (H3 T11ph) is induced by meiotic DSBs in and Molecular genetic experiments in confirmed that Mek1 is required for H3 T11ph and revealed that phosphorylation is rapidly reversed when Mek1 kinase is no longer active. Reconstituting histone phosphorylation with recombinant proteins demonstrated that Mek1 directly catalyzes H3 T11 phosphorylation. Mutating H3 T11 to nonphosphorylatable residues conferred no detectable defects in otherwise unperturbed meiosis, although the mutations modestly reduced spore viability in certain strains where Rad51 is used for strand exchange in place of Dmc1. H3 T11ph is therefore mostly dispensable for Mek1 function. However, H3 T11ph provides an excellent marker of ongoing Mek1 kinase activity Anti-H3 T11ph chromatin immunoprecipitation followed by deep sequencing demonstrated that H3 T11ph was highly enriched at presumed sites of attachment of chromatin to chromosome axes, gave a more modest signal along chromatin loops, and was present at still lower levels immediately adjacent to DSB hotspots. These localization patterns closely tracked the distribution of Red1 and Hop1, axis proteins required for Mek1 activation. These findings provide insight into the spatial disposition of Mek1 kinase activity and the higher order organization of recombining meiotic chromosomes.

No abstract

Covalent post-translational modifications of histones have important functions in transcription, replication, repair, and other aspects of eukaryotic chromosome dynamics. Trimethylation of lysine-4 on histone H3 is enriched at actively transcribed loci in many organisms. The impact of this histone modification on transcription has been extensively studied, but less is known about its effects on other chromosomal processes. An intriguing new study in this

The 2.0 Å crystal structure has been determined for Escherichia coli uridine phosphorylase (UP), an essential enzyme in nucleotide biosynthesis that catalyzes the phosphorolytic cleavage of the C—N glycosidic bond of uridine to ribose‐1‐phosphate and uracil. The structure determination of two independent monomers in the asymmetric unit revealed the residue composition and atomic details of the apo configurations of each active site. The native hexameric UP enzyme was revealed by applying threefold crystallographic symmetry to the contents of the asymmetric unit. The 2.0 Å model reveals a closer structural relationship to other nucleotide phosphorylase enzymes than was previously appreciated.