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Nature Apr 2024RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs). However, the mechanism by which...
RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs). However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.
Topics: Humans; Cryoelectron Microscopy; DNA; DNA Breaks, Double-Stranded; DNA Repair; Nucleosomes; Protein Subunits; Rad51 Recombinase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Mutation; Protein Domains; Histones; Protein Binding
PubMed: 38509361
DOI: 10.1038/s41586-024-07196-4 -
Frontiers in Cell and Developmental... 2024
PubMed: 38500684
DOI: 10.3389/fcell.2024.1386038 -
Nucleic Acids Research May 2024Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have...
Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage. Here, we successfully prepared highly pure A. thaliana MTOPVIB and MTOPVIB-SPO11 complex. Contrary to expectations, our findings highlight that MTOPVIB differs from orthologous Topo VIB by lacking ATP-binding activity and independently forming dimers without ATP. Most significantly, our study reveals that while MTOPVIB lacks the capability to stimulate SPO11-mediated DNA cleavage, it functions as a bona fide DNA-binding protein and plays a substantial role in facilitating the dsDNA binding capacity of the MOTOVIB-SPO11 complex. Thus, we illustrate mechanistic divergence between the MTOPVIB-SPO11 complex and classical type IIB topoisomerases.
Topics: Adenosine Triphosphate; Arabidopsis; Arabidopsis Proteins; Archaeal Proteins; DNA Breaks, Double-Stranded; DNA Topoisomerases; DNA Topoisomerases, Type II; DNA-Binding Proteins; Endodeoxyribonucleases; Evolution, Molecular; Meiosis; Protein Multimerization
PubMed: 38499490
DOI: 10.1093/nar/gkae181 -
Nucleus (Austin, Tex.) Dec 2024Heterochromatin is an organizational property of eukaryotic chromosomes, characterized by extensive DNA and histone modifications, that is associated with the silencing... (Review)
Review
Heterochromatin is an organizational property of eukaryotic chromosomes, characterized by extensive DNA and histone modifications, that is associated with the silencing of transposable elements and repetitive sequences. Maintaining heterochromatin is crucial for ensuring genomic integrity and stability during the cell cycle. During meiosis, heterochromatin is important for homologous chromosome synapsis, recombination, and segregation, but our understanding of meiotic heterochromatin formation and condensation is limited. In this review, we focus on the dynamics and features of heterochromatin and how it condenses during meiosis in plants. We also discuss how meiotic heterochromatin influences the interaction and recombination of homologous chromosomes during prophase I.
Topics: Heterochromatin; Centromere; Meiosis; Chromosome Pairing
PubMed: 38488152
DOI: 10.1080/19491034.2024.2328719 -
Cellular & Molecular Biology Letters Mar 2024DNA mismatch repair (MMR) is a highly conserved pathway that corrects DNA replication errors, the loss of which is attributed to the development of various types of...
BACKGROUND
DNA mismatch repair (MMR) is a highly conserved pathway that corrects DNA replication errors, the loss of which is attributed to the development of various types of cancers. Although well characterized, MMR factors remain to be identified. As a 3'-5' exonuclease and endonuclease, meiotic recombination 11 homolog A (MRE11A) is implicated in multiple DNA repair pathways. However, the role of MRE11A in MMR is unclear.
METHODS
Initially, short-term and long-term survival assays were used to measure the cells' sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Meanwhile, the level of apoptosis was also determined by flow cytometry after MNNG treatment. Western blotting and immunofluorescence assays were used to evaluate the DNA damage within one cell cycle after MNNG treatment. Next, a GFP-heteroduplex repair assay and microsatellite stability test were used to measure the MMR activities in cells. To investigate the mechanisms, western blotting, the GFP-heteroduplex repair assay, and chromatin immunoprecipitation were used.
RESULTS
We show that knockdown of MRE11A increased the sensitivity of HeLa cells to MNNG treatment, as well as the MNNG-induced DNA damage and apoptosis, implying a potential role of MRE11 in MMR. Moreover, we found that MRE11A was largely recruited to chromatin and negatively regulated the DNA damage signals within the first cell cycle after MNNG treatment. We also showed that knockdown of MRE11A increased, while overexpressing MRE11A decreased, MMR activity in HeLa cells, suggesting that MRE11A negatively regulates MMR activity. Furthermore, we show that recruitment of MRE11A to chromatin requires MLH1 and that MRE11A competes with PMS2 for binding to MLH1. This decreases PMS2 levels in whole cells and on chromatin, and consequently comprises MMR activity.
CONCLUSIONS
Our findings reveal that MRE11A is a negative regulator of human MMR.
Topics: Humans; Chromatin; DNA Mismatch Repair; HeLa Cells; Methylnitronitrosoguanidine; Mismatch Repair Endonuclease PMS2
PubMed: 38486171
DOI: 10.1186/s11658-024-00547-z -
Nature Communications Mar 2024The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a...
The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a conserved membrane fluidity sensor, AdipoR2, regulates the meiosis-specific lipidome in mouse testes by promoting the synthesis of sphingolipids containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs). AdipoR2 upregulates the expression of a fatty acid elongase, ELOVL2, both transcriptionally and post-transcriptionally, to synthesize VLC-PUFA. The depletion of VLC-PUFAs and subsequent accumulation of palmitic acid in AdipoR2 knockout testes stiffens the cellular membrane and causes the invagination of the nuclear envelope. This condition impairs the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Further, the stiffened membrane impairs the formation of intercellular bridges and the germ cell syncytium, which disrupts the orderly arrangement of cell types within the seminiferous tubules. According to our findings we propose a framework in which the highly-fluid membrane microenvironment shaped by AdipoR2-ELOVL2 underpins meiosis-specific chromosome dynamics in testes.
Topics: Animals; Male; Mice; Carrier Proteins; Meiosis; Membrane Fluidity; Nuclear Envelope; Sphingolipids; Telomere
PubMed: 38485951
DOI: 10.1038/s41467-024-46718-6 -
BioRxiv : the Preprint Server For... Mar 2024HELB is a human helicase involved in initiation of DNA replication, the replication stress response, and regulation of double-strand DNA break repair. rs75770066 is a...
HELB is a human helicase involved in initiation of DNA replication, the replication stress response, and regulation of double-strand DNA break repair. rs75770066 is a rare SNP in the HELB gene that affects age at natural menopause. rs75770066 results in a D506G substitution in an acidic patch within the 1A domain of the helicase that is known to interact with RPA. We found that this amino acid change dramatically impairs the cellular function of HELB. D506G-HELB exhibits impaired interaction with RPA, which likely results in the effects of rs75770066 as this reduces recruitment of HELB to sites of DNA damage. Reduced recruitment of D506G-HELB to double-strand DNA breaks and the concomitant increase in homologous recombination likely alters the levels of meiotic recombination, which affects the viability of gametes. Because menopause occurs when oocyte levels drop below a minimum threshold, altered repair of meiotic double-stranded DNA breaks has the potential to directly affect the age at natural menopause.
PubMed: 38464108
DOI: 10.1101/2024.02.27.582415 -
Science Advances Mar 2024Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of...
Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of premature cell division, where nonreplicated, G/G-stage somatic cell nuclei are transplanted to the metaphase cytoplasm of mouse oocytes. Subsequent cell division generates daughter cells with reduced ploidy. Unexpectedly, genome sequencing analysis revealed proper segregation of homologous chromosomes, resulting in complete haploid genomes. We observed a high occurrence of somatic genome haploidization in nuclei from inbred genetic backgrounds but not in hybrids, emphasizing the importance of sequence homology between homologs. These findings suggest that premature cell division relies on mechanisms similar to meiosis I, where genome haploidization is facilitated by homologous chromosome interactions, recognition, and pairing. Unlike meiosis, no evidence of recombination between somatic cell homologs was detected. Our study offers an alternative in vitro gametogenesis approach by directly reprogramming diploid somatic cells into haploid oocytes.
Topics: Animals; Mice; Haploidy; Diploidy; Meiosis; Cell Nucleus; Chromatids
PubMed: 38457500
DOI: 10.1126/sciadv.adk9001 -
Plant Molecular Biology Mar 2024Knowing how chromosome recombination works is essential for plant breeding. It enables the design of crosses between different varieties to combine desirable traits and...
Knowing how chromosome recombination works is essential for plant breeding. It enables the design of crosses between different varieties to combine desirable traits and create new ones. This is because the meiotic crossovers between homologous chromatids are not purely random, and various strategies have been developed to describe and predict such exchange events. Recent studies have used methylation data to predict chromosomal recombination in rice using machine learning models. This approach proved successful due to the presence of a positive correlation between the CHH context cytosine methylation and recombination rates in rice chromosomes. This paper assesses the question if methylation can be used to predict recombination in four plant species: Arabidopsis, maize, sorghum, and tomato. The results indicate a positive association between CHH context methylation and recombination rates in certain plant species, with varying degrees of strength in their relationships. The CG and CHG methylation contexts show negative correlation with recombination. Methylation data was key effectively in predicting recombination in sorghum and tomato, with a mean determination coefficient of 0.65 ± 0.11 and 0.76 ± 0.05, respectively. In addition, the mean correlation values between predicted and experimental recombination rates were 0.83 ± 0.06 for sorghum and 0.90 ± 0.05 for tomato, confirming the significance of methylomes in both monocotyledonous and dicotyledonous species. The predictions for Arabidopsis and maize were not as accurate, likely due to the comparatively weaker relationships between methylation contexts and recombination, in contrast to sorghum and tomato, where stronger associations were observed. To enhance the accuracy of predictions, further evaluations using data sets closely related to each other might prove beneficial. In general, this methylome-based method holds great potential as a reliable strategy for predicting recombination rates in various plant species, offering valuable insights to breeders in their quest to develop novel and improved varieties.
Topics: Arabidopsis; Epigenome; Plant Breeding; DNA Methylation; Plants; Recombination, Genetic; Gene Expression Regulation, Plant
PubMed: 38457042
DOI: 10.1007/s11103-023-01396-8 -
Cell Discovery Mar 2024Single-cell whole-genome sequencing methods have undergone great improvements over the past decade. However, allele dropout, which means the inability to detect both...
Single-cell whole-genome sequencing methods have undergone great improvements over the past decade. However, allele dropout, which means the inability to detect both alleles simultaneously in an individual diploid cell, largely restricts the application of these methods particularly for medical applications. Here, we develop a new single-cell whole-genome sequencing method based on third-generation sequencing (TGS) platform named Refresh-seq (restriction fragment ligation-based genome amplification and TGS). It is based on restriction endonuclease cutting and ligation strategy in which two alleles in an individual cell can be cut into equal fragments and tend to be amplified simultaneously. As a new single-cell long-read genome sequencing method, Refresh-seq features much lower allele dropout rate compared with SMOOTH-seq. Furthermore, we apply Refresh-seq to 688 sperm cells and 272 female haploid cells (secondary polar bodies and parthenogenetic oocytes) from F1 hybrid mice. We acquire high-resolution genetic map of mouse meiosis recombination at low sequencing depth and reveal the sexual dimorphism in meiotic crossovers. We also phase the structure variations (deletions and insertions) in sperm cells and female haploid cells with high precision. Refresh-seq shows great performance in screening aneuploid sperm cells and oocytes due to the low allele dropout rate and has great potential for medical applications such as preimplantation genetic diagnosis.
PubMed: 38443370
DOI: 10.1038/s41421-023-00638-9