The Role of AtMUS81 in Interference-Insensitive Crossovers in A. thaliana

MUS81 is conserved among plants, animals, and fungi and is known to be involved in mitotic DNA damage repair and meiotic recombination. Here we present a functional characterization of the Arabidopsis thaliana homolog AtMUS81, which has a role in both mitotic and meiotic cells. The AtMUS81 transcript is produced in all tissues, but is elevated greater than 9-fold in the anthers and its levels are increased in response to gamma radiation and methyl methanesulfonate treatment. An Atmus81 transfer-DNA insertion mutant shows increased sensitivity to a wide range of DNA-damaging agents, confirming its role in mitotically proliferating cells. To examine its role in meiosis, we employed a pollen tetrad–based visual assay. Data from genetic intervals on Chromosomes 1 and 3 show that Atmus81 mutants have a moderate decrease in meiotic recombination. Importantly, measurements of recombination in a pair of adjacent intervals on Chromosome 5 demonstrate that the remaining crossovers in Atmus81 are interference sensitive, and that interference levels in the Atmus81 mutant are significantly greater than those in wild type. These data are consistent with the hypothesis that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive

Analyzing Somatic DNA Repair in Arabidopsis Meiotic Mutants

Meiotic and somatic recombination share a common set of factors. Thus, the analysis of somatic DNA repair in meiotic mutant lines should be of special interest. Growth defects of mutant plants induced by specific genotoxins can thereby hint to DNA repair functions of the affected proteins. Here, we describe two kinds of approaches to characterize deficiencies in DNA repair in mutant lines of Arabidopsis thaliana, after genotoxin treatment

The CYCLIN-A CYCA1;2/TAM Is Required for the Meiosis I to Meiosis II Transition and Cooperates with OSD1 for the Prophase to First Meiotic Division Transition

Meiosis halves the chromosome number because its two divisions follow a single round of DNA replication. This process involves two cell transitions, the transition from prophase to the first meiotic division (meiosis I) and the unique meiosis I to meiosis II transition. We show here that the A-type cyclin CYCA1;2/TAM plays a major role in both transitions in Arabidopsis. A series of tam mutants failed to enter meiosis II and thus produced diploid spores and functional diploid gametes. These diploid gametes had a recombined genotype produced through the single meiosis I division. In addition, by combining the tam-2 mutation with AtSpo11-1 and Atrec8, we obtained plants producing diploid gametes through a mitotic-like division that were genetically identical to their parents. Thus tam alleles displayed phenotypes very similar to that of the previously described osd1 mutant. Combining tam and osd1 mutations leads to a failure in the prophase to meiosis I transition during male meiosis and to the production of tetraploid spores and gametes. This suggests that TAM and OSD1 are involved in the control of both meiotic transitions

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastroph

Genomic Structure of and Genome-Wide Recombination in the Saccharomyces cerevisiae S288C Progenitor Isolate EM93

The diploid isolate EM93 is the main ancestor to the widely used Saccharomyces cerevisiae haploid laboratory strain, S288C. In this study, we generate a high-resolution overview of the genetic differences between EM93 and S288C. We show that EM93 is heterozygous for >45,000 polymorphisms, including large sequence polymorphisms, such as deletions and a Saccharomyces paradoxus introgression. We also find that many large sequence polymorphisms (LSPs) are associated with Ty-elements and sub-telomeric regions. We identified 2,965 genetic markers, which we then used to genotype 120 EM93 tetrads. In addition to deducing the structures of all EM93 chromosomes, we estimate that the average EM93 meiosis produces 144 detectable recombination events, consisting of 87 crossover and 31 non-crossover gene conversion events. Of the 50 polymorphisms showing the highest levels of non-crossover gene conversions, only three deviated from parity, all of which were near heterozygous LSPs. We find that non-telomeric heterozygous LSPs significantly reduce meiotic recombination in adjacent intervals, while sub-telomeric LSPs have no discernable effect on recombination. We identified 203 recombination hotspots, relatively few of which are hot for both non-crossover gene conversions and crossovers. Strikingly, we find that recombination hotspots show limited conservation. Some novel hotspots are found adjacent to heterozygous LSPs that eliminate other hotspots, suggesting that hotspots may appear and disappear relatively rapidly

Analysis of Biological Features Associated with Meiotic Recombination Hot and Cold Spots in Saccharomyces cerevisiae

Meiotic recombination is not distributed uniformly throughout the genome. There are regions of high and low recombination rates called hot and cold spots, respectively. The recombination rate parallels the frequency of DNA double-strand breaks (DSBs) that initiate meiotic recombination. The aim is to identify biological features associated with DSB frequency. We constructed vectors representing various chromatin and sequence-based features for 1179 DSB hot spots and 1028 DSB cold spots. Using a feature selection approach, we have identified five features that distinguish hot from cold spots in Saccharomyces cerevisiae with high accuracy, namely the histone marks H3K4me3, H3K14ac, H3K36me3, and H3K79me3; and GC content. Previous studies have associated H3K4me3, H3K36me3, and GC content with areas of mitotic recombination. H3K14ac and H3K79me3 are novel predictions and thus represent good candidates for further experimental study. We also show nucleosome occupancy maps produced using next generation sequencing exhibit a bias at DSB hot spots and this bias is strong enough to obscure biologically relevant information. A computational approach using feature selection can productively be used to identify promising biological associations. H3K14ac and H3K79me3 are novel predictions of chromatin marks associated with meiotic DSBs. Next generation sequencing can exhibit a bias that is strong enough to lead to incorrect conclusions. Care must be taken when interpreting high throughput sequencing data where systematic biases have been documented

Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes

Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81–Mms4/EME1, Slx1–Slx4/BTBD12/MUS312, XPF–ERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination