RNase H enables efficient repair of R-loop induced DNA damage.

R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. We present a model that the persistence of R-loops at sites of DNA damage induces repair by break-induced replication (BIR). This R-loop induced BIR is particularly susceptible to the formation of lethal repair intermediates at the rDNA because of a barrier imposed by RNA polymerase I

Use of a distant reporter group as evidence for a conformational change in a sensory receptor

A highly sensitive method for demonstrating ligand-induced conformational changes in protein molecules in solution is described. The method utilizes an environmentally sensitive reporter group that is known to be distant from the active site. In the present application a conformational change is demonstrated in the galactose receptor of Salmonella typhimurium, involved in bacterial sensing and transport, by means of an extrinsic fluorophore, 5-iodoacetamidofluorescein, attached at a single methionine residue, and the intrinsic tryptophan fluorophore. Binding of the ligand galactose perturbs the microenvironment of both the fluorescein and tryptophan, as shown by both spectral and potassium iodide quenching changes. The distance between the two dyes is established by fluorescence energy transfer methods to be 41 ± 10 angstrom. Since only one molecule of galactose binds per molecule of receptor and since the galactose molecule is only about 5 angstrom in length, changes at one of these sites reflect the result of an indirect effect. Hence, there must be a ligand-induced conformational change that is propagated a minimum of 30 angstrom through the receptor molecule

In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin

The machinery mediating chromosome condensation is poorly understood. To begin to dissect the in vivo function(s) of individual components, we monitored mitotic chromosome structure in mutants of condensin, cohesin, histone H3, and topoisomerase II (topo II). In budding yeast, both condensation establishment and maintenance require all of the condensin subunits, but not topo II activity or phospho-histone H3. Structural maintenance of chromosome (SMC) protein 2, as well as each of the three non-SMC proteins (Ycg1p, Ycs4p, and Brn1p), was required for chromatin binding of the condensin complex in vivo. Using reversible condensin alleles, we show that chromosome condensation does not involve an irreversible modification of condensin or chromosomes. Finally, we provide the first evidence of a mechanistic link between condensin and cohesin function. A model discussing the functional interplay between cohesin and condensin is presented

Genetic drift at expanding frontiers promotes gene segregation

Competition between random genetic drift and natural selection plays a central role in evolution: Whereas non-beneficial mutations often prevail in small populations by chance, mutations that sweep through large populations typically confer a selective advantage. Here, however, we observe chance effects during range expansions that dramatically alter the gene pool even in large microbial populations. Initially well-mixed populations of two fluorescently labeled strains of Escherichia coli develop well-defined, sector-like regions with fractal boundaries in expanding colonies. The formation of these regions is driven by random fluctuations that originate in a thin band of pioneers at the expanding frontier. A comparison of bacterial and yeast colonies (Saccharomyces cerevisiae) suggests that this large-scale genetic sectoring is a generic phenomenon that may provide a detectable footprint of past range expansions.Comment: Please visit http://www.pnas.org/content/104/50/19926.abstract for published articl

Precise Asymptotics for a Random Walker's Maximum

We consider a discrete time random walk in one dimension. At each time step the walker jumps by a random distance, independent from step to step, drawn from an arbitrary symmetric density function. We show that the expected positive maximum E[M_n] of the walk up to n steps behaves asymptotically for large n as, E[M_n]/\sigma=\sqrt{2n/\pi}+ \gamma +O(n^{-1/2}), where \sigma^2 is the variance of the step lengths. While the leading \sqrt{n} behavior is universal and easy to derive, the leading correction term turns out to be a nontrivial constant \gamma. For the special case of uniform distribution over [-1,1], Coffmann et. al. recently computed \gamma=-0.516068...by exactly enumerating a lengthy double series. Here we present a closed exact formula for \gamma valid for arbitrary symmetric distributions. We also demonstrate how \gamma appears in the thermodynamic limit as the leading behavior of the difference variable E[M_n]-E[|x_n|] where x_n is the position of the walker after n steps. An application of these results to the equilibrium thermodynamics of a Rouse polymer chain is pointed out. We also generalize our results to L\'evy walks.Comment: new references added, typos corrected, published versio

Engineering of Cyclodextrin Product Specificity and pH Optima of the Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 β-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-Å resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced α-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (β- and γ-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of α-CD. Glu258 is involved in catalysis in CGTases as well as α-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.

Substrate-Assisted Catalysis Unifies Two Families of Chitinolytic Enzymes

Hen egg-white lysozyme has long been the paradigm for enzymatic glycosyl hydrolysis with retention of configuration, with a protonated carboxylic acid and a deprotonated carboxylate participating in general acid-base catalysis. In marked contrast, the retaining chitin degrading enzymes from glycosyl hydrolase families 18 and 20 all have a single glutamic acid as the catalytic acid but lack a nucleophile on the enzyme. Both families have a catalytic (βα)8-barrel domain in common. X-ray structures of three different chitinolytic enzymes complexed with substrates or inhibitors identify a retaining mechanism involving a protein acid and the carbonyl oxygen atom of the substrate’s C2 N-acetyl group as the nucleophile. These studies unambiguously demonstrate the distortion of the sugar ring toward a sofa conformation, long postulated as being close to that of the transition state in glycosyl hydrolysis.

Area distribution and the average shape of a L\'evy bridge

We consider a one dimensional L\'evy bridge x_B of length n and index 0 < \alpha < 2, i.e. a L\'evy random walk constrained to start and end at the origin after n time steps, x_B(0) = x_B(n)=0. We compute the distribution P_B(A,n) of the area A = \sum_{m=1}^n x_B(m) under such a L\'evy bridge and show that, for large n, it has the scaling form P_B(A,n) \sim n^{-1-1/\alpha} F_\alpha(A/n^{1+1/\alpha}), with the asymptotic behavior F_\alpha(Y) \sim Y^{-2(1+\alpha)} for large Y. For \alpha=1, we obtain an explicit expression of F_1(Y) in terms of elementary functions. We also compute the average profile < \tilde x_B (m) > at time m of a L\'evy bridge with fixed area A. For large n and large m and A, one finds the scaling form = n^{1/\alpha} H_\alpha({m}/{n},{A}/{n^{1+1/\alpha}}), where at variance with Brownian bridge, H_\alpha(X,Y) is a non trivial function of the rescaled time m/n and rescaled area Y = A/n^{1+1/\alpha}. Our analytical results are verified by numerical simulations.Comment: 21 pages, 4 Figure