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Fly Apr 2017Somatic recombination is essential to protect genomes of somatic cells from DNA damage but it also has important clinical implications, as it is a driving force of...
Somatic recombination is essential to protect genomes of somatic cells from DNA damage but it also has important clinical implications, as it is a driving force of tumorigenesis leading to inactivation of tumor suppressor genes. Despite this importance, our knowledge about somatic recombination in adult tissues remains very limited. Our recent work, using the Drosophila adult midgut has demonstrated that spontaneous events of mitotic recombination accumulate in aging adult intestinal stem cells and result in frequent loss of heterozygosity (LOH). In this Extra View article, we provide further data supporting long-track chromosome LOH and discuss potential mechanisms involved in the process. In addition, we further discuss relevant questions surrounding somatic recombination and how the mechanisms and factors influencing somatic recombination in adult tissues can be explored using the Drosophila midgut model.
Topics: Adult Stem Cells; Animals; Clonal Evolution; Drosophila; Intestines; Loss of Heterozygosity; Mitosis; Models, Animal; Recombination, Genetic
PubMed: 27834607
DOI: 10.1080/19336934.2016.1249073 -
The Journal of Cell Biology Jun 2008Centromeres are special structures of eukaryotic chromosomes that hold sister chromatid together and ensure proper chromosome segregation during cell division....
Centromeres are special structures of eukaryotic chromosomes that hold sister chromatid together and ensure proper chromosome segregation during cell division. Centromeres consist of repeated sequences, which have hindered the study of centromere mitotic recombination and its consequences for centromeric function. We use a chromosome orientation fluorescence in situ hybridization technique to visualize and quantify recombination events at mouse centromeres. We show that centromere mitotic recombination occurs in normal cells to a higher frequency than telomere recombination and to a much higher frequency than chromosome-arm recombination. Furthermore, we show that centromere mitotic recombination is increased in cells lacking the Dnmt3a and Dnmt3b DNA methyltransferases, suggesting that the epigenetic state of centromeric heterochromatin controls recombination events at these regions. Increased centromere recombination in Dnmt3a,3b-deficient cells is accompanied by changes in the length of centromere repeats, suggesting that prevention of illicit centromere recombination is important to maintain centromere integrity in the mouse.
Topics: Animals; Centromere; Chromosomes, Mammalian; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA, Satellite; Embryonic Stem Cells; Genotype; In Situ Hybridization, Fluorescence; Mice; Mice, Inbred C57BL; Minisatellite Repeats; Mitosis; Recombination, Genetic; Sister Chromatid Exchange; Telomere
PubMed: 18541703
DOI: 10.1083/jcb.200803042 -
PLoS Genetics Mar 2021Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in...
Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3' single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we show that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.
Topics: Animals; Apoptosis; BRCA2 Protein; Crossing Over, Genetic; DNA End-Joining Repair; DNA-Directed DNA Polymerase; Drosophila melanogaster; Gene Expression Regulation; Holliday Junction Resolvases; Homologous Recombination; Mitosis; Synthetic Lethal Mutations; DNA Polymerase theta
PubMed: 33750946
DOI: 10.1371/journal.pgen.1009267 -
Molecular Cell Aug 2019Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the...
Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid (mitosis) or enforce (meiosis) the formation of reciprocal exchanges-crossovers-between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity purified 7 DNA-processing enzymes that channel HR intermediates into crossovers or noncrossovers from vegetative cells or cells undergoing meiosis. Using mass spectrometry, we provide a global characterization of their composition and reveal mitosis- and meiosis-specific modules in the interaction networks. Functional analyses of meiosis-specific interactors of MutLγ-Exo1 identified Rtk1, Caf120, and Chd1 as regulators of crossing-over. Chd1, which transiently associates with Exo1 at the prophase-to-metaphase I transition, enables the formation of MutLγ-dependent crossovers through its conserved ability to bind and displace nucleosomes. Thus, rewiring of the HR network, coupled to chromatin remodeling, promotes context-specific control of the recombination outcome.
Topics: Crossing Over, Genetic; Mass Spectrometry; Meiosis; Mitosis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31351878
DOI: 10.1016/j.molcel.2019.06.022 -
BMC Bioinformatics Nov 2021Identifying haplotypes is central to sequence analysis in diploid or polyploid genomes. Despite this, there remains a lack of research and tools designed for physical...
BACKGROUND
Identifying haplotypes is central to sequence analysis in diploid or polyploid genomes. Despite this, there remains a lack of research and tools designed for physical phasing and its downstream analysis.
RESULTS
HaplotypeTools is a new toolset to phase variant sites using VCF and BAM files and to analyse phased VCFs. Phasing is achieved via the identification of reads overlapping ≥ 2 heterozygous positions and then extended by additional reads, a process that can be parallelized across a computer cluster. HaplotypeTools includes various utility scripts for downstream analysis including crossover detection and phylogenetic placement of haplotypes to other lineages or species. HaplotypeTools was assessed for accuracy against WhatsHap using simulated short and long reads, demonstrating higher accuracy, albeit with reduced haplotype length. HaplotypeTools was also tested on real Illumina data to determine the ancestry of hybrid fungal isolate Batrachochytrium dendrobatidis (Bd) SA-EC3, finding 80% of haplotypes across the genome phylogenetically cluster with parental lineages BdGPL (39%) and BdCAPE (41%), indicating those are the parental lineages. Finally, ~ 99% of phasing was conserved between overlapping phase groups between SA-EC3 and either parental lineage, indicating mitotic gene conversion/parasexuality as the mechanism of recombination for this hybrid isolate. HaplotypeTools is open source and freely available from https://github.com/rhysf/HaplotypeTools under the MIT License.
CONCLUSIONS
HaplotypeTools is a powerful resource for analyzing hybrid or recombinant diploid or polyploid genomes and identifying parental ancestry for sub-genomic regions.
Topics: Algorithms; Genomics; Haplotypes; High-Throughput Nucleotide Sequencing; Phylogeny; Polymorphism, Single Nucleotide; Recombination, Genetic; Sequence Analysis, DNA
PubMed: 34809571
DOI: 10.1186/s12859-021-04473-1 -
Microbiology and Molecular Biology... Jun 1999The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past... (Comparative Study)
Comparative Study Review
The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.
Topics: Adenosine Triphosphatases; Animals; DNA Damage; DNA Helicases; DNA Repair; DNA Repair Enzymes; DNA Replication; DNA, Fungal; DNA-Binding Proteins; Deoxyribonucleases, Type II Site-Specific; Fungal Proteins; Humans; Meiosis; Rad52 DNA Repair and Recombination Protein; Rec A Recombinases; Recombination, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 10357855
DOI: 10.1128/MMBR.63.2.349-404.1999 -
Nucleic Acids Research Mar 2010RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early... (Review)
Review
RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA-binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or co-mediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic re-stabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51 expression.
Topics: Animals; DNA Repair; Genes, p53; Genomic Instability; Humans; Models, Genetic; Mutation; Neoplasms; Rad51 Recombinase; Recombination, Genetic
PubMed: 19942681
DOI: 10.1093/nar/gkp1063 -
Anais Da Academia Brasileira de Ciencias Dec 2014Mitotic recombination is a process involved in carcinogenesis which can lead to genetic loss through the loss of heterozygosity. The recombinogenic potentials of two...
Mitotic recombination is a process involved in carcinogenesis which can lead to genetic loss through the loss of heterozygosity. The recombinogenic potentials of two anticancer drugs topoisomerase I inhibitors, camptothecin (CPT) and irinotecan (CPT-11), were evaluated in the present study. The homozygotization assay, which assess the induction of mitotic recombination and gene homozygosis, as well as the heterozygous A757//UT448 diploid strain of Aspergillus nidulans were employed. The three non-cytotoxic concentrations of CPT (3.5 ng mL-1, 10.5 ng mL-1 and 17.4 ng mL-1) were found to induce both mitotic recombination and gene homozygosis. CPT treatment produced three diploids homozygous, for nutritional and conidia color genes, and Homozygotization Indices (HI) significantly different from negative control. On the other hand, only the highest CPT-11 concentration tested (18 µg mL-1), corresponding to the maximal single chemotherapeutic dose, produced HI values higher than 2.0 and significantly different from negative control HI values. The recombinogenic effects of both topoisomerase I blockers were associated with the recombinational repair of DNA strand breaks induced by CPT and CPT-11. The anticancer drugs CPT and CPT-11 may be characterized as secondary malignancies promoters in cancer patients after chemotherapy treatment.
Topics: Aspergillus nidulans; Camptothecin; Diploidy; Homozygote; Irinotecan; Mitosis; Mutagenicity Tests; Recombination, Genetic; Topoisomerase I Inhibitors
PubMed: 25590709
DOI: 10.1590/0001-3765201420130106 -
DNA Repair Aug 2017Double-strand breaks (DSBs) are among the most lethal DNA lesions, and a variety of pathways have evolved to manage their repair in a timely fashion. One such pathway is... (Review)
Review
Double-strand breaks (DSBs) are among the most lethal DNA lesions, and a variety of pathways have evolved to manage their repair in a timely fashion. One such pathway is homologous recombination (HR), in which information from an undamaged donor site is used as a template for repair. Although many of the biochemical steps of HR are known, the physical movements of chromosomes that must underlie the pairing of homologous sequence during mitotic DSB repair have remained mysterious. Recently, several groups have begun to use a variety of genetic and cell biological tools to study this important question. These studies reveal that both damaged and undamaged loci increase the volume of the nuclear space that they explore after the formation of DSBs. This DSB-induced increase in chromosomal mobility is regulated by many of the same factors that are important during HR, such as ATR-dependent checkpoint activation and the recombinase Rad51, suggesting that this phenomenon may facilitate the search for homology. In this perspective, we review current research into the mobility of chromosomal loci during HR, as well as possible underlying mechanisms, and discuss the critical questions that remain to be answered. Although we focus primarily on recent studies in the budding yeast, Saccharomyces cerevisiae, examples of experiments performed in higher eukaryotes are also included, which reveal that increased mobility of damaged loci is a process conserved throughout evolution.
Topics: Chromosomes; DNA; DNA Breaks, Double-Stranded; Eukaryota; Recombinational DNA Repair; Saccharomyces cerevisiae
PubMed: 28663070
DOI: 10.1016/j.dnarep.2017.06.012 -
Microbiology and Molecular Biology... Dec 2002The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote... (Review)
Review
The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.
Topics: Animals; Crossing Over, Genetic; DNA Repair; DNA-Binding Proteins; Epistasis, Genetic; Humans; Recombination, Genetic; Saccharomyces cerevisiae
PubMed: 12456786
DOI: 10.1128/MMBR.66.4.630-670.2002