scholarly journals Recombination-independent recognition of DNA homology for meiotic silencing inNeurospora crassa

2019 ◽  
Author(s):  
Nicholas Rhoades ◽  
Germano Cecere ◽  
Thomas Hammond ◽  
Eugene Gladyshev
Keyword(s):  
Meiotic Pairing ◽  
Small Interfering Rnas ◽  
Dna Breaks ◽  
Meiotic Silencing ◽  
Base Pairs ◽  
Homologous Chromosomes ◽  
Meiotic Chromosomes ◽  
Double Strand Dna Breaks ◽  
Dna Fragment ◽  
Inneurospora Crassa

ABSTRACTPairing of homologous chromosomes represents a key aspect of meiosis in nearly all sexually reproducing species. While meiotic pairing requires the formation of double-strand DNA breaks in some organisms, in many others it can proceed in the apparent absence of DNA breakage and recombination. The mechanistic nature of such recombination-independent pairing represents a fundamental question in molecular biology. Using “meiotic silencing by unpaired DNA” (MSUD) in the fungusNeurospora crassaas a model system, we demonstrate the existence of a cardinally new solution to the problem of inter-chromosomal homology recognition during meiosis. Here we take advantage of the unique ability of MSUD to efficiently detect and silence (by RNA interference) any relatively short DNA fragment lacking a homologous allelic partner. We show that MSUD does not require the function of eukaryotic RecA proteins and the type II topoisomerase-like protein Spo11. We further show that MSUD can recognize patterns of weak interspersed homology in which short units of sequence identity are arrayed with a periodicity of 11 base-pairs (bp). Taken together, these results reveal the function of a recombination-independent homology-directed process in guiding the expression of small interfering RNAs and suggest that meiotic chromosomes can be evaluated for sequence homology at base-pair resolution by a mechanism that operates on intact DNA molecules.

scholarly journals Recombination-independent recognition of DNA homology for meiotic silencing in Neurospora crassa

2021 ◽  
Vol 118 (33) ◽  
pp. e2108664118
Author(s):  
Nicholas Rhoades ◽  
Tinh-Suong Nguyen ◽  
Guillaume Witz ◽  
Germano Cecere ◽  
Thomas Hammond ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Dna Homology ◽  
Meiotic Silencing ◽  
Base Pairs ◽  
Homologous Chromosomes ◽  
Sequence Identity ◽  
Experimental Systems ◽  
Double Stranded Dna ◽  
Dna Fragment ◽  
Homology Recognition

The pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. In many organisms, pairing involves chromosomes that remain apparently intact. The mechanistic nature of homology recognition at the basis of such pairing is unknown. Using “meiotic silencing by unpaired DNA” (MSUD) as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here, we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesion complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be colinear and must contain short tracts of sequence identity spaced apart at 21 or 22 base pairs. By using these periodicity values as a guiding parameter in all-atom molecular modeling, we discover that homologous DNA molecules can pair by forming quadruplex-based contacts with an interval of 2.5 helical turns. This process requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. Our results 1) reconcile genetic and biophysical evidence for the existence of direct homologous double-stranded DNA (dsDNA)–dsDNA pairing, 2) identify a role for this process in initiating RNA interference, and 3) suggest that chromosomes can be cross-matched by a precise mechanism that operates on intact dsDNA molecules.

Timing of molecular events in meiosis in Saccharomyces cerevisiae: stable heteroduplex DNA is formed late in meiotic prophase

1993 ◽  
Vol 13 (1) ◽  
pp. 373-382 ◽  
Author(s):  
C Goyon ◽  
M Lichten
Keyword(s):  
Meiotic Prophase ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
Base Pairs ◽  
Heteroduplex Dna ◽  
Strand Breaks ◽  
Dna Structures ◽  
Double Strand Dna Breaks ◽  
Molecular Events ◽  
The Times

To better understand the means by which chromosomes pair and recombine during meiosis, we have determined the time of appearance of heteroduplex DNA relative to the times of appearance of double-strand DNA breaks and of mature recombined molecules. Site-specific double-strand breaks appeared early in meiosis and were formed and repaired with a timing consistent with a role for breaks as initiators of recombination. Heteroduplex-containing molecules appeared about 1 h after double-strand breaks and were followed shortly by crossover products and the first meiotic nuclear division. We conclude that parental chromosomes are stably joined in heteroduplex-containing structures late in meiotic prophase and that these structures are rapidly resolved to yield mature crossover products. If the chromosome pairing and synapsis observed earlier in meiotic prophase is mediated by formation of biparental DNA structures, these structures most likely either contain regions of non-Watson-Crick base pairs or contain regions of heteroduplex DNA that either are very short or dissociate during DNA purification. Two loci were examined in this study: the normal ARG4 locus, and an artificial locus consisting of an arg4-containing plasmid inserted at MAT. Remarkably, sequences in the ARG4 promoter that suffered double-strand cleavage at the normal ARG4 locus were not cut at significant levels when present at MAT::arg4. These results indicate that the formation of double-strand breaks during meiosis does not simply involve the specific recognition and cleavage of a short nucleotide sequence.

scholarly journals Mer2 binds directly to both nucleosomes and axial proteins as the keystone of meiotic recombination

2020 ◽  
Author(s):  
Dorota Rousova ◽  
Saskia K. Funk ◽  
Heidi Reichle ◽  
John R. Weir
Keyword(s):  
Sexual Reproduction ◽  
Meiotic Recombination ◽  
Dna Breaks ◽  
Homologous Chromosomes ◽  
Protein Biochemistry ◽  
Double Strand Dna Breaks ◽  
Domain Protein ◽  
Meiosis I ◽  
Double Strand Dna

One of the defining features of sexual reproduction is the recombination events that take place during meiosis I. Recombination is both evolutionarily advantageous, but also mechanistically necessary to form the crossovers that link homologous chromosomes. Meiotic recombination is initiated through the placement of programmed double-strand DNA breaks (DSBs) mediated by the protein Spo11. The timing, number, and physical placement of DSBs are carefully controlled through a variety of protein machinery. Previous work has implicated Mer2(IHO1 in mammals) to be involved in both the placement of breaks, and their timing. In this study we use a combination of protein biochemistry and biophysics to extensively characterise various roles of the Mer2. We gain further insights into the details of Mer2 interaction with the PHD protein Spp1, reveal that Mer2 is a novel nucleosome binder, and suggest how Mer2’s interaction with the HORMA domain protein Hop1 (HORMAD1/2 in mammals) is controlled.

scholarly journals Timing of molecular events in meiosis in Saccharomyces cerevisiae: stable heteroduplex DNA is formed late in meiotic prophase.

1993 ◽  
Vol 13 (1) ◽  
pp. 373-382 ◽  
Author(s):  
C Goyon ◽  
M Lichten
Keyword(s):  
Meiotic Prophase ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
Base Pairs ◽  
Heteroduplex Dna ◽  
Strand Breaks ◽  
Dna Structures ◽  
Double Strand Dna Breaks ◽  
Molecular Events ◽  
The Times

To better understand the means by which chromosomes pair and recombine during meiosis, we have determined the time of appearance of heteroduplex DNA relative to the times of appearance of double-strand DNA breaks and of mature recombined molecules. Site-specific double-strand breaks appeared early in meiosis and were formed and repaired with a timing consistent with a role for breaks as initiators of recombination. Heteroduplex-containing molecules appeared about 1 h after double-strand breaks and were followed shortly by crossover products and the first meiotic nuclear division. We conclude that parental chromosomes are stably joined in heteroduplex-containing structures late in meiotic prophase and that these structures are rapidly resolved to yield mature crossover products. If the chromosome pairing and synapsis observed earlier in meiotic prophase is mediated by formation of biparental DNA structures, these structures most likely either contain regions of non-Watson-Crick base pairs or contain regions of heteroduplex DNA that either are very short or dissociate during DNA purification. Two loci were examined in this study: the normal ARG4 locus, and an artificial locus consisting of an arg4-containing plasmid inserted at MAT. Remarkably, sequences in the ARG4 promoter that suffered double-strand cleavage at the normal ARG4 locus were not cut at significant levels when present at MAT::arg4. These results indicate that the formation of double-strand breaks during meiosis does not simply involve the specific recognition and cleavage of a short nucleotide sequence.

scholarly journals Synaptonemal complex proteins direct and constrain the localization of crossover-promoting proteins during Caenorhabditis elegans meiosis

2019 ◽  
Author(s):  
Cori K. Cahoon ◽  
Jacquellyn M. Helm ◽  
Diana E. Libuda
Keyword(s):  
Caenorhabditis Elegans ◽  
Synaptonemal Complex ◽  
Meiotic Chromosome ◽  
Variable Number ◽  
Specific Structure ◽  
Dna Breaks ◽  
Late Prophase ◽  
Homologous Chromosomes ◽  
Sister Chromatids ◽  
Double Strand Dna Breaks

AbstractCrossovers (COs) between homologous chromosomes are critical for meiotic chromosome segregation and form in the context of the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologs. During Caenorhabditis elegans meiosis, central region components of the SC (SYP proteins) are essential to repair double-strand DNA breaks (DSBs) as COs, but the roles of these SYP proteins in promoting CO formation are poorly understood. Here, we investigate the relationships between the SYP proteins and conserved CO-promoting factors by examining the immunolocalization of these factors in meiotic mutants where SYP proteins are absent, reduced, or mis-localized. Although COs do not form in syp null mutants, CO-promoting proteins COSA-1, MSH-5, and ZHP-3 nevertheless become co-localized at a variable number of DSB-dependent sites during late prophase, reflecting an inherent affinity of these factors for DSB repair sites. In contrast, in mutants where SYP proteins are present but form aggregates or display abnormal synapsis, CO-promoting proteins consistently track with SYP-1 localization. Moreover, CO-promoting proteins usually localize to a single site per SYP-1 structure, even in SYP aggregates or in mutants where SC forms between sister-chromatids, suggesting that CO regulation occurs within these structures. Further, we find that sister chromatids in the meiotic cohesin mutant rec-8 require both CO-promoting proteins and the SC to remain connected. Taken together, our findings support a model in which SYP proteins promote CO formation by directing and constraining the localization of CO-promoting factors to ensure that CO maturation occurs only between properly aligned homologous chromosomes.Article SummaryErrors during meiosis are the leading cause of birth defects and miscarriages in humans. Thus, the coordinated control of meiosis events is critical for the faithful inheritance of the genome each generation. The synaptonemal complex (SC) is a meiosis-specific structure that assembles between homologs chromosomes and is critical for the establishment and regulation of crossovers, which ensure the accurate segregation of the homologous chromosomes at meiosis I. Here we show that the SC proteins function to regulate crossovers by directing and constraining the localization of proteins involved in promoting the formation of crossovers.

scholarly journals Nuclear myosin/actin-motored contact between homologous chromosomes is initiated by ATM kinase and homology-directed repair proteins at double-strand DNA breaks to suppress chromosome rearrangements

Oncotarget ◽  
2018 ◽  
Vol 9 (17) ◽  
pp. 13612-13622 ◽  
Author(s):  
Viktoria N. Evdokimova ◽  
Manoj Gandhi ◽  
Alyaksandr V. Nikitski ◽  
Christopher J. Bakkenist ◽  
Yuri E. Nikiforov
Keyword(s):  
Chromosome Rearrangements ◽  
Dna Breaks ◽  
Atm Kinase ◽  
Homologous Chromosomes ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Double Strand Dna

scholarly journals The Escherichia coli Methyl-Directed Mismatch Repair System Repairs Base Pairs Containing Oxidative Lesions

2003 ◽  
Vol 185 (5) ◽  
pp. 1701-1704 ◽  
Author(s):  
Jennifer Wyrzykowski ◽  
Michael R. Volkert
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Spontaneous Mutation ◽  
Repair System ◽  
Dna Breaks ◽  
Base Pairs ◽  
Double Strand Dna Breaks ◽  
Hydrogen Peroxide Treatment ◽  
Mismatch Repair System ◽  
Dna Strands

ABSTRACT A major role of the methyl-directed mismatch repair (MMR) system of Escherichia coli is to repair postreplicative errors. In this report, we provide evidence that MMR also acts on oxidized DNA, preventing mutagenesis. When cells deficient in MMR are grown anaerobically, spontaneous mutation frequencies are reduced compared with those of the same cells grown aerobically. In addition, we show that a dam mutant has an increased sensitivity to hydrogen peroxide treatment that can be suppressed by mutations that inactivate MMR. In a dam mutant, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted to single- and double-strand DNA breaks. Thus, base pairs containing oxidized bases will be converted to strand breaks if they are repaired by MMR. This is demonstrated by the increased peroxide sensitivity of a dam mutant and the finding that the sensitivity can be suppressed by mutations inactivating MMR. We demonstrate further that this repair activity results from MMR recognition of base pairs containing 8-oxoguanine (8-oxoG) based on the finding that overexpression of the MutM oxidative repair protein, which repairs 8-oxoG, can suppress the mutH-dependent increase in transversion mutations. These findings demonstrate that MMR has the ability to prevent oxidative mutagenesis either by removing 8-oxoG directly or by removing adenine misincorporated opposite 8-oxoG or both.

scholarly journals Control of meiotic pairing and recombination by chromosomally tethered 26S proteasome

Science ◽  
2017 ◽  
Vol 355 (6323) ◽  
pp. 408-411 ◽  
Author(s):  
Jasvinder S. Ahuja ◽  
Rima Sandhu ◽  
Rana Mainpal ◽  
Crystal Lawson ◽  
Hanna Henley ◽  
...  
Keyword(s):  
Synaptonemal Complex ◽  
Double Strand Breaks ◽  
26S Proteasome ◽  
Strand Exchange ◽  
Crossing Over ◽  
Meiotic Pairing ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Meiotic Chromosomes ◽  
Evolutionarily Conserved

During meiosis, paired homologous chromosomes (homologs) become linked via the synaptonemal complex (SC) and crossovers. Crossovers mediate homolog segregation and arise from self-inflicted double-strand breaks (DSBs). Here, we identified a role for the proteasome, the multisubunit protease that degrades proteins in the nucleus and cytoplasm, in homolog juxtaposition and crossing over. Without proteasome function, homologs failed to pair and instead remained associated with nonhomologous chromosomes. Although dispensable for noncrossover formation, a functional proteasome was required for a coordinated transition that entails SC assembly between longitudinally organized chromosome axes and stable strand exchange of crossover-designated DSBs. Notably, proteolytic core and regulatory proteasome particles were recruited to chromosomes by Zip3, the ortholog of mammalian E3 ligase RNF212, and SC protein Zip1 . We conclude that proteasome functions along meiotic chromosomes are evolutionarily conserved.

Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

2019 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Cellular Response ◽  
Sequencing Data ◽  
Dna Breaks ◽  
Precise Integration ◽  
Guide Rna ◽  
Double Strand Dna Breaks ◽  
Human Therapy ◽  
P53 Activation ◽  
Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

scholarly journals Fine-Structure Mapping of Meiosis-Specific Double-Strand DNA Breaks at a Recombination Hotspot Associated With an Insertion of Telomeric Sequences Upstream of the HIS4 Locus in Yeast

Genetics ◽  
1996 ◽  
Vol 143 (3) ◽  
pp. 1115-1125 ◽  
Author(s):  
Fei Xu ◽  
Thomas D Petes
Keyword(s):  
Meiotic Recombination ◽  
Hydroxyl Groups ◽  
Sequence Specificity ◽  
Structure Mapping ◽  
Dna Breaks ◽  
Exonuclease Iii ◽  
Recombination Hotspot ◽  
Telomeric Sequences ◽  
Double Strand Dna Breaks ◽  
Double Strand Dna

Abstract Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs). Using two approaches, we mapped the position of DSBs associated with a recombination hotspot created by insertion of telomeric sequences into the region upstream of HIS4. We found that the breaks have no obvious sequence specificity and localize to a region of ~50 bp adjacent to the telomeric insertion. By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3′-hydroxyl groups.

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