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Genetics Mar 2008The mismatch repair (MMR) system is critical not only for the repair of DNA replication errors, but also for the regulation of mitotic and meiotic recombination...
The mismatch repair (MMR) system is critical not only for the repair of DNA replication errors, but also for the regulation of mitotic and meiotic recombination processes. In a manner analogous to its ability to remove replication errors, the MMR system can remove mismatches in heteroduplex recombination intermediates to generate gene conversion events. Alternatively, such mismatches can trigger an MMR-dependent antirecombination activity that blocks the completion of recombination, thereby limiting interactions between diverged sequences. In Saccharomyces cerevisiae, the MMR proteins Msh3, Msh6, and Mlh1 interact with proliferating cell nuclear antigen (PCNA), and mutations that disrupt these interactions result in a mutator phenotype. In addition, some mutations in the PCNA-encoding POL30 gene increase mutation rates in an MMR-dependent manner. In the current study, pol30, mlh1, and msh6 mutants were used to examine whether MMR-PCNA interactions are similarly important during mitotic and meiotic recombination. We find that MMR-PCNA interactions are important for repairing mismatches formed during meiotic recombination, but play only a relatively minor role in regulating the fidelity of mitotic recombination.
Topics: Alleles; Chromosome Segregation; DNA Mismatch Repair; Meiosis; Mitosis; Mutagenesis; Mutation; Nucleic Acid Heteroduplexes; Proliferating Cell Nuclear Antigen; Protein Binding; Recombination, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 18245822
DOI: 10.1534/genetics.107.085415 -
Molecular and Cellular Biology Jan 1991The effect of the strong promoter from the alcohol dehydrogenase gene on mitotic and meiotic intragenic recombination has been studied at the ade6 locus of the fission...
The effect of the strong promoter from the alcohol dehydrogenase gene on mitotic and meiotic intragenic recombination has been studied at the ade6 locus of the fission yeast Schizosaccharomyces pombe. A 700-bp fragment containing the functional adh1 promoter was used to replace the weak wild-type promoter of the ade6 gene. Analysis of mRNA showed that strains with this ade6::adh1 fusion construct had strongly elevated ade6-specific mRNA levels during vegetative growth as well as in meiosis. These increased levels of mRNA correlated with a 20- to 25-fold stimulation of intragenic recombination in meiosis and a 7-fold increased prototroph formation during vegetative growth. Analysis of flanking marker configurations of prototrophic recombinants indicated that simple conversions as well as conversions associated with crossing over were stimulated in meiosis. The strongest stimulation of recombination was observed when the adh1 promoter was homozygous. Studies with heterologous promoter configurations revealed that the highly transcribed allele was the preferred acceptor of genetic information. The effect of the recombinational hot spot mutation ade6-M26 was also investigated in this system. Its effect was only partly additive to the elevated recombination rate generated by the ade6::adh1 fusion construct.
Topics: Alcohol Dehydrogenase; DNA, Fungal; Gene Conversion; Meiosis; Mitosis; Promoter Regions, Genetic; RNA, Fungal; RNA, Messenger; Recombination, Genetic; Restriction Mapping; Schizosaccharomyces; Transcription, Genetic
PubMed: 1986226
DOI: 10.1128/mcb.11.1.289-298.1991 -
Proceedings of the National Academy of... Aug 1991Loss of heterozygosity is a significant oncogenetic mechanism and can involve a variety of mechanisms including chromosome loss, deletion, and homologous...
Loss of heterozygosity is a significant oncogenetic mechanism and can involve a variety of mechanisms including chromosome loss, deletion, and homologous interchromosomal mitotic recombination. Analysis of H-2 antigen-loss variants from heterozygous murine cell lines provides an experimental system to estimate the relative contributions of different mechanisms for allele loss and to compare the chromosomal patterns of mitotic and meiotic recombination. Cytotoxic anti-H-2D antibodies and complement were used to isolate 161 independent target antigen-negative clones from H-2d/H-2b heterozygous cell lines; of these, 131 (84.5%) lost the allele encoding the target antigen. Allele-loss variants were typed and scored as either heterozygous or homozygous for six H-2D-proximal chromosome 17 markers and for one distal marker by restriction enzyme-site variations and Southern analysis. A single mitotic crossover could account for 50 clones (37%), with heterozygosity for at least one proximal marker and loss of heterozygosity for all markers distal to the putative recombination site. Eighty-two allele-loss variants (60%) were homozygous for all markers; the origin of these clones could be either chromosome loss or mitotic recombination between the centromere and the most proximal marker. Only 4 clones (3%) arose through more complex events such as multiple crossovers or deletion. A mitotic linkage map for mouse chromosome 17 was constructed, and the gene order deduced from somatic recombination was identical to that obtained by conventional transmission genetics. These results demonstrate that mitotic recombination is a common event leading to allele loss, in spite of the lack of evidence for frequent somatic pairing of homologous chromosomes. Mitotic mapping provides a defined system for comparison of mitotic and meiotic recombination and may lead to practical advances for elucidating somatic mechanisms of oncogenesis and for gene therapy in targeting mutations to specific sites through homologous recombination.
Topics: Alleles; Animals; Base Sequence; Cell Line; Chromosome Mapping; Crosses, Genetic; DNA Probes; Female; Genetic Linkage; Genetic Variation; H-2 Antigens; Heterozygote; Homozygote; Male; Meiosis; Mice; Mice, Inbred Strains; Mitosis; Molecular Sequence Data; Oligonucleotide Probes; Polymorphism, Restriction Fragment Length; Recombination, Genetic
PubMed: 1677769
DOI: 10.1073/pnas.88.15.6486 -
Development (Cambridge, England) Dec 2013Gene targeting by 'ends-out' homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision...
Gene targeting by 'ends-out' homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR.
Topics: Animals; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Breaks, Double-Stranded; Drosophila melanogaster; Gene Targeting; Genetic Markers; Genetic Vectors; Homologous Recombination; Mutagenesis, Insertional; Recombination, Genetic; Sequence Deletion
PubMed: 24154526
DOI: 10.1242/dev.100933 -
PLoS Genetics Nov 2013Mammalian common fragile sites are loci of frequent chromosome breakage and putative recombination hotspots. Here, we utilized Replication Slow Zones (RSZs), a budding...
Mammalian common fragile sites are loci of frequent chromosome breakage and putative recombination hotspots. Here, we utilized Replication Slow Zones (RSZs), a budding yeast homolog of the mammalian common fragile sites, to examine recombination activities at these loci. We found that rates of URA3 inactivation of a hisG-URA3-hisG reporter at RSZ and non-RSZ loci were comparable under all conditions tested, including those that specifically promote chromosome breakage at RSZs (hydroxyurea [HU], mec1Δ sml1Δ, and high temperature), and those that suppress it (sml1Δ and rrm3Δ). These observations indicate that RSZs are not recombination hotspots and that chromosome fragility and recombination activity can be uncoupled. Results confirmed recombinogenic effects of HU, mec1Δ sml1Δ, and rrm3Δ and identified temperature as a regulator of mitotic recombination. We also found that these conditions altered the nature of recombination outcomes, leading to a significant increase in the frequency of URA3 inactivation via loss of heterozygosity (LOH), the type of genetic alteration involved in cancer development. Further analyses revealed that the increase was likely due to down regulation of intrachromatid and intersister (IC/IS) bias in mitotic recombination, and that RSZs exhibited greater sensitivity to HU dependent loss of IC/IS bias than non RSZ loci. These observations suggest that recombinogenic conditions contribute to genome rearrangements not only by increasing the overall recombination activity, but also by altering the nature of recombination outcomes by their effects on recombination partner choice. Similarly, fragile sites may contribute to cancer more frequently than non-fragile loci due their enhanced sensitivity to certain conditions that down-regulate the IC/IS bias rather than intrinsically higher rates of recombination.
Topics: Chromosome Breakage; Chromosome Fragile Sites; DNA Replication; Hydroxyurea; Mitosis; Mutation; Recombination, Genetic; Saccharomyces cerevisiae
PubMed: 24244194
DOI: 10.1371/journal.pgen.1003931 -
Genetics Jul 1988Allelic and nonallelic (ectopic) recombination events were analyzed in a set of isogenic strains that carry marked Ty elements. We found that allelic recombination...
Allelic and nonallelic (ectopic) recombination events were analyzed in a set of isogenic strains that carry marked Ty elements. We found that allelic recombination between Ty elements occurred at normal frequencies both in meiosis and mitosis. The marked Ty elements were involved in a large variety of different types of ectopic recombination and this variety was greater in mitosis than in meiosis. Allelic and ectopic recombination events occurred at similar frequencies in mitosis, but allelic recombination predominated in meiosis. Some of the types of ectopic mitotic recombination indicated the common occurrence of concerted recombination events. The length of homology represented by a delta element (330 bp) seemed to be sufficient for some types of mitotic and meiotic recombination.
Topics: Alleles; DNA Transposable Elements; Genes, Fungal; Meiosis; Mitosis; Plasmids; Recombination, Genetic; Saccharomyces cerevisiae
PubMed: 2841187
DOI: 10.1093/genetics/119.3.549 -
The Journal of Biological Chemistry Jan 2018It has been long assumed that post-mitotic neurons only utilize the error-prone non-homologous end-joining pathway to repair double-strand breaks (DSBs) associated with...
It has been long assumed that post-mitotic neurons only utilize the error-prone non-homologous end-joining pathway to repair double-strand breaks (DSBs) associated with oxidative damage to DNA, given the inability of non-replicating neuronal DNA to utilize a sister chromatid template in the less error-prone homologous recombination (HR) repair pathway. However, we and others have found recently that active transcription triggers a replication-independent recombinational repair mechanism in G/G phase of the cell cycle. Here we observed that the HR repair protein RAD52 is recruited to sites of DNA DSBs in terminally differentiated, post-mitotic neurons. This recruitment is dependent on the presence of a nascent mRNA generated during active transcription, providing evidence that an RNA-templated HR repair mechanism exists in non-dividing, terminally differentiated neurons. This recruitment of RAD52 in neurons is decreased by transcription inhibition. Importantly, we found that high concentrations of amyloid β, a toxic protein associated with Alzheimer's disease, inhibits the expression and DNA damage response of RAD52, potentially leading to a defect in the error-free, RNA-templated HR repair mechanism. This study shows a novel RNA-dependent repair mechanism of DSBs in post-mitotic neurons and demonstrates that defects in this pathway may contribute to neuronal genomic instability and consequent neurodegenerative phenotypes such as those seen in Alzheimer's disease.
Topics: Animals; DNA Breaks, Double-Stranded; G1 Phase; Mitosis; Neurons; RNA; Rad52 DNA Repair and Recombination Protein; Rats; Recombination, Genetic; Resting Phase, Cell Cycle
PubMed: 29217771
DOI: 10.1074/jbc.M117.808402 -
Genetic Testing and Molecular Biomarkers Jan 2012The aims of the present study were to assess (1) the parental origin of trisomy 21 and the stage in which nondisjunction occurs and (2) the relationship between altered...
The aims of the present study were to assess (1) the parental origin of trisomy 21 and the stage in which nondisjunction occurs and (2) the relationship between altered genetic recombination and maternal age as risk factors for trisomy 21. The study included 102 cases with Down syndrome from the Croatian population. Genotyping analyses were performed by polymerase chain reaction using 11 short tandem repeat markers along chromosome 21q. The vast majority of trisomy 21 was of maternal origin (93%), followed by paternal (5%) and mitotic origin (2%). The frequencies of maternal meiotic I (MI) and meiotic II errors were 86% and 14%, respectively. The highest proportion of cases with zero recombination was observed among those with maternal MI derived trisomy 21. A higher proportion of telomeric exchanges were presented in cases with maternal MI errors and cases with young mothers, although these findings were not statistically significant. The present study is the first report examining parental origin and altered genetic recombination as a risk factor for trisomy 21 in a Croatian population. The results support that trisomy 21 has a universal genetic etiology across different human populations.
Topics: Adolescent; Adult; Croatia; Disease Susceptibility; Down Syndrome; Female; Humans; Inheritance Patterns; Male; Maternal Age; Meiosis; Nondisjunction, Genetic; Parents; Pregnancy; Recombination, Genetic; Risk Factors; Young Adult
PubMed: 21861707
DOI: 10.1089/gtmb.2011.0066 -
Genetics Oct 2018Under neutrality, linkage disequilibrium results from physically linked sites having nonindependent coalescent histories. In obligately sexual organisms, meiotic...
Under neutrality, linkage disequilibrium results from physically linked sites having nonindependent coalescent histories. In obligately sexual organisms, meiotic recombination is the dominant force separating linked variants from one another, and thus in determining the decay of linkage disequilibrium with physical distance. In facultatively sexual diploid organisms that principally reproduce clonally, mechanisms of mitotic exchange are expected to become relatively more important in shaping linkage disequilibrium. Here we outline mathematical and computational models of a facultative-sex coalescent process that includes meiotic and mitotic recombination, via both crossovers and gene conversion, to determine how linkage disequilibrium is affected with facultative sex. We demonstrate that the degree to which linkage disequilibrium is broken down by meiotic recombination simply scales with the probability of sex if it is sufficiently high (much greater than [Formula: see text] for population size ). However, with very rare sex (occurring with frequency on the order of [Formula: see text]), mitotic gene conversion plays a particularly important and complicated role because it both breaks down associations between sites and removes within-individual diversity. Strong population structure under rare sex leads to lower average linkage disequilibrium values than in panmictic populations, due to the influence of low-frequency polymorphisms created by allelic sequence divergence acting in individual subpopulations. These analyses provide information on how to interpret observed linkage disequilibrium patterns in facultative sexuals and to determine what genomic forces are likely to shape them.
Topics: Animals; Diploidy; Female; Gene Conversion; Linkage Disequilibrium; Male; Models, Genetic; Sex
PubMed: 30097538
DOI: 10.1534/genetics.118.301244 -
Nucleic Acids Research Jun 1992Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments...
Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments in vivo by meiotic recombination. This is of importance for reconstruction of a long gene or a gene complex. In this work we have taken advantage of yeast protoplast fusion to generate isosexual diploids followed by mitotic crossing-over, and show that it can be an alternative simple strategy for recombining YACs. Integrative transformation of one of the parent strains with the construct pRAN4 (containing the ADE2 gene) is used to disrupt the URA3 gene contained within the pYAC4 vector arm, providing the markers required for forcing fusion and detecting recombination. All steps can be carried out within the commonly used AB1380 host strain without the requirement for micromanipulation. The method was applied to YAC clones from the human MHC and resulted in the reconstruction of a 650 kb long single clone containing 18 known genes from the MHC class II region.
Topics: Blotting, Southern; Chromosomes, Fungal; Cloning, Molecular; Genes, MHC Class II; Humans; Mitosis; Plasmids; Recombination, Genetic; Restriction Mapping; Yeasts
PubMed: 1620611
DOI: 10.1093/nar/20.12.3135