The kinetochore prevents centromere-proximal crossover recombination during meiosis

During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. DOI: http://dx.doi.org/10.7554/eLife.10850.00

ReCombine: A Suite of Programs for Detection and Analysis of Meiotic Recombination in Whole-Genome Datasets

In meiosis, the exchange of DNA between chromosomes by homologous recombination is a critical step that ensures proper chromosome segregation and increases genetic diversity. Products of recombination include reciprocal exchanges, known as crossovers, and non-reciprocal gene conversions or non-crossovers. The mechanisms underlying meiotic recombination remain elusive, largely because of the difficulty of analyzing large numbers of recombination events by traditional genetic methods. These traditional methods are increasingly being superseded by high-throughput techniques capable of surveying meiotic recombination on a genome-wide basis. Next-generation sequencing or microarray hybridization is used to genotype thousands of polymorphic markers in the progeny of hybrid yeast strains. New computational tools are needed to perform this genotyping and to find and analyze recombination events. We have developed a suite of programs, ReCombine, for using short sequence reads from next-generation sequencing experiments to genotype yeast meiotic progeny. Upon genotyping, the program CrossOver, a component of ReCombine, then detects recombination products and classifies them into categories based on the features found at each location and their distribution among the various chromatids. CrossOver is also capable of analyzing segregation data from microarray experiments or other sources. This package of programs is designed to allow even researchers without computational expertise to use high-throughput, whole-genome methods to study the molecular mechanisms of meiotic recombination

Aspects of Steel Structures in Marine Environment including Corrosion Using Eurocode 3 and National Annex

Steel structures in the marine environment like Steel Sheet Pile walls at quays and harbors, undergo continuous corrosion subjected to prolong sea water environment. The design of these steel structures currently is been practiced by using respective national annexure in alliance with the Eurocodes. But the correct statistical properties which fit into the semi-probabilistic safety concept of the Eurocodes still have to be determined. Steel Sheet pile retaining walls will obligatorily be analyzed according to the Eurocodes on and after the year 2010 [1]. Eurocodes 1 and 3 for load assumptions and structural steel design are introduced to various countries by building authorities even now. The work on Eurocodes 1997-1:2004 has been recently completed; to focus on the harmonization of all the national annexures and an economical design of steel sheet piles must guaranteed remain. The current paper highlights mainly upon the difference in the design aspects with special consideration to the usage of partial safety factor dealt in the Eurocodes, versus using initial practice of the respective national annexure and various geotechnical factors affecting the same. Analytical examples of typical cross sections of sheet piles will be evaluated using annexure and in conjunction with Eurocode and FEM packages

Data from: Controlling meiotic recombinational repair: specifying the roles of ZMMs, Sgs1 and Mus81/Mms4 in crossover formation

Crossovers (COs) play a critical role in ensuring proper alignment and segregation of homologous chromosomes during meiosis. How the cell balances recombination between CO vs. noncrossover (NCO) outcomes is not completely understood. Further lacking is what constrains the extent of DNA repair such that multiple events do not arise from a single double-strand break (DSB). Here, by interpreting signatures that result from recombination genome-wide, we find that synaptonemal complex proteins promote crossing over in distinct ways. Our results suggest that Zip3 (RNF212) promotes biased cutting of the double Holliday-junction (dHJ) intermediate whereas surprisingly Msh4 does not. Moreover, detailed examination of conversion tracts in sgs1 and mms4-md mutants reveal distinct aberrant recombination events involving multiple chromatid invasions. In sgs1 mutants, these multiple invasions are generally multichromatid involving 3–4 chromatids; in mms4-md mutants the multiple invasions preferentially resolve into one or two chromatids. Our analysis suggests that Mus81/Mms4 (Eme1), rather than just being a minor resolvase for COs is crucial for both COs and NCOs in preventing chromosome entanglements by removing 3′- flaps to promote second-end capture. Together our results force a reevaluation of how key recombination enzymes collaborate to specify the outcome of meiotic DNA repair

Data from: Reduced crossover interference and increased ZMM-independent recombination in the absence of Tel1/ATM

Meiotic recombination involves the repair of double-strand break (DSB) precursors as crossovers (COs) or noncrossovers (NCOs). The proper number and distribution of COs is critical for successful chromosome segregation and formation of viable gametes. In budding yeast the majority of COs occurs through a pathway dependent on the ZMM proteins (Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3), which form foci at CO-committed sites. Here we show that the DNA-damage-response kinase Tel1/ATM limits ZMM-independent recombination. By whole-genome mapping of recombination products, we find that lack of Tel1 results in higher recombination and reduced CO interference. Yet the number of Zip3 foci in tel1Δ cells is similar to wild type, and these foci show normal interference. Analysis of recombination in a tel1Δ zip3Δ double mutant indicates that COs are less dependent on Zip3 in the absence of Tel1. Together these results reveal that in the absence of Tel1, a significant proportion of COs occurs through a non-ZMM-dependent pathway, contributing to a CO landscape with poor interference. We also see a significant change in the distribution of all detectable recombination products in the absence of Tel1, Sgs1, Zip3, or Msh4, providing evidence for altered DSB distribution. These results support the previous finding that DSB interference depends on Tel1, and further suggest an additional level of DSB interference created through local repression of DSBs around CO-designated sites

PGENETICS_ZMM_SGS1_MMS4_ReCombine_data

PGENETICS_ZMM_SGS1_MMS4_ReCombine_dat

Data from: High throughput sequencing reveals alterations in the recombination signatures with diminishing spo11 activity

Spo11 is the topoisomerase-like enzyme responsible for the induction of the meiosis-specific double strand breaks (DSBs), which initiate the recombination events responsible for proper chromosome segregation. Nineteen PCR-induced alleles of SPO11 were identified and characterized genetically and cytologically. Recombination, spore viability and synaptonemal complex (SC) formation were decreased to varying extents in these mutants. Arrest by ndt80 restored these events in two severe hypomorphic mutants, suggesting that ndt80-arrested nuclei are capable of extended DSB activity. While crossing-over, spore viability and synaptonemal complex (SC) formation defects correlated, the extent of such defects was not predictive of the level of heteroallelic gene conversions (prototrophs) exhibited by each mutant. High throughput sequencing of tetrads from spo11 hypomorphs revealed that gene conversion tracts associated with COs are significantly longer and gene conversion tracts unassociated with COs are significantly shorter than in wild type. By modeling the extent of these tract changes, we could account for the discrepancy in genetic measurements of prototrophy and crossover association. These findings provide an explanation for the unexpectedly low prototroph levels exhibited by spo11 hypomorphs and have important implications for genetic studies that assume an unbiased recovery of prototrophs, such as measurements of CO homeostasis. Our genetic and physical data support previous observations of DSB-limited meioses, in which COs are disproportionally maintained over NCOs (CO homeostasis)

Distribution of GCco and NCO tract lengths for <i>sgs1</i> and <i>zmm</i> mutants.

<p>(A) Comparison of GCco vs. NCO tract lengths for WT (B) Comparison of GCco vs. NCO tract lengths for <i>sgs1</i>. (C) NCO length distributions for <i>sgs1</i>, <i>msh4sgs1</i> and <i>zip3sgs1</i> (D) Box plot of GC_CO lengths. The dark line in each box represents median GC_CO length. Each box outlines lower and upper quartile. The whiskers denote the boundaries of the interquartile range and the circles outside the whiskers represent outliers (outside 1.5 times the interquartile range). The value inside each box is the median GC_CO length. (E) Comparison of GC_CO vs. NCO tract lengths for <i>msh4</i> (F) Comparison of GC_CO vs. NCO tract lengths for <i>zip3</i>.</p

Model of the relationship between Sgs1, Zip3 and Msh4 in CO-NCO fate.

<p>Models for how (A) WT, (B) <i>zip3</i> and (C) <i>sgs1</i> might produce the distribution of observed recombination types. Indicated in bold type are the main signatures observed. Arrow weight indicates the relative degree of partitioning through the pathway. Expected biased or unbiased cuts are indicated by asterisks or filled arrowheads.</p