Biology subjects

Neale, M. J.

Publications and source records attributed to Neale, M. J..

12 recordsLinked to original sources

bioRxiv2026

Transcription directs Holliday junction branch migration

During meiosis, genetic diversity arises from the resolution of branched DNA intermediates called Holliday junctions to create crossovers--sites of reciprocal exchange between parental chromosomes. Holliday junctions arise during the repair of Spo11-induced DNA breaks, yet the principles linking break formation, repair, and chromosome architecture remain unclear. Top3, a type IA topoisomerase, acts on Holliday junctions, but its spatiotemporal dynamics are unknown. Here, we map Top3 catalytic activity throughout meiotic prophase with strand specificity and nucleotide resolution. We identify a DNA sequence motif associated with catalysis and a pattern of activity around Spo11 hotspots that requires ongoing repair and Top3s helicase partner, Sgs1. Strikingly, Top3 activity shifts over time, influenced by transcription, cohesin, and the crossover factors Msh5 and Mer3, redistributing toward sites of convergent transcription--known locations of meiotic cohesin association. Upon prophase exit, as crossovers are resolved, Top3 activity subsides. Remarkably, maps of genome-wide recombination reveal that crossover resolution preferentially occurs at these same regions of convergent transcription. Collectively, we propose that Top3 coordinates the transcription-coupled movement of Holliday junctions from Spo11 hotspots towards cohesin-associated axis sites, whereupon crossover resolution occurs to ensure accurate meiotic chromosome segregation.

molecular biology↗

bioRxiv2026

Localised negative feedback shapes genome-wide patterning of meiotic DNA breaks

Genetic diversity within sexually reproducing species arises via the formation and repair of programmed DNA double-strand breaks (DSBs) created by the evolutionarily conserved topoisomerase-like enzyme, Spo11. Because DSBs threaten genome stability, their formation is tightly regulated in both space and time. In S. cerevisiae, Tel1, the orthologue of mammalian Ataxia Telangiectasia Mutated (ATM) kinase, suppresses nearby DSB formation through local inhibition known as DSB interference. However, whether such local inhibition reshapes the genome-wide DSB landscape remains unclear. Here, we develop a quantitative simulation framework to model how Tel1-mediated feedback shapes Spo11-DSB formation across the yeast genome. We demonstrate that innate chromosome-specific DSB patterns, when combined with interference, generate complex, population-level redistribution of DSBs. We define the spatial range over which interference propagates and provide evidence that this regulatory mechanism requires Tel1 recruitment to DSBs via Xrs2 and Tel1 kinase activity. Although the pro-DSB factor Rec114 contributes to DSB regulation, mutation of potential Rec114 phosphorylation sites indicates that it is not an essential target of Tel1. Together, these findings demonstrate how localised negative feedback can drive broad-scale, emergent patterning of a fundamental genome-modifying process, with the potential in meiosis to influence recombination initiation and, consequently, genetic variation across generations.

cell biology↗

bioRxiv2023

Hi-C2B: Optimised detection of chromosomal contacts within synchronised meiotic S. cerevisiae cells

Hi-C, a genome-wide chromosome conformation capture assay is a powerful tool used to study three-dimensional genome organisation by converting physical pairwise interactions into counts of pairwise interaction. To study the many temporally regulated facets of meiotic recombination in S. cerevisiae the Hi-C assay must be robust such that fine- and wide-scale comparisons between genetic datasets can be made. Here we describe an updated protocol for Hi-C (Hi-C2B) that generates reproducible libraries of interaction data with low noise and for a relatively low cost.

molecular biology↗

bioRxiv2023

CC-seq: Nucleotide-resolution mapping of Spo11 DNA double-strand breaks in S. cerevisiae cells

i.During meiosis Spo11 generates DNA double-strand breaks to induce recombination, becoming covalently attached to the 5' ends on both sides of the break during this process. Such Spo11 "covalent complexes" are transient in wild-type cells, but accumulate in nuclease mutants unable to initiate repair. The CC-seq method presented here details how to map the location of these Spo11 complexes genome-wide with strand-specific nucleotide-resolution accuracy in synchronised Saccharomyces cerevisiae meiotic cells.

molecular biology↗

bioRxiv2023

Inhibition of Topoisomerase 2 catalytic activity impacts the integrity of heterochromatin and repetitive DNA and leads to interlinks between clustered repeats

DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an Slx4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements.

cell biology↗

bioRxiv2023

Meiotic prophase length modulates Tel1-dependent DNA double-strand break interference

During meiosis, genetic recombination is initiated by the formation of many DNA double-strand breaks (DSBs) catalysed by the evolutionarily conserved topoisomerase-like enzyme, Spo11, in preferred genomic sites known as hotspots. DSB formation activates the Tel1/ATM DNA damage responsive (DDR) kinase, locally inhibiting Spo11 activity in adjacent hotspots via a process known as DSB interference. Intriguingly, in S. cerevisiae, over short genomic distances (<15 kb), Spo11 activity displays characteristics of concerted activity or clustering, wherein the frequency of DSB formation in adjacent hotspots is greater than expected by chance. We have proposed that clustering is caused by a limited number of sub-chromosomal domains becoming primed for DSB formation. Here, we demonstrate that DSB clustering is abolished when meiotic prophase timing is extended via deletion of the NDT80 transcription factor. We propose that extension of meiotic prophase enables most cells, and therefore most chromosomal domains within them, to reach an equilibrium state of similar Spo11-DSB potential, reducing the impact that priming has on estimates of coincident DSB formation. Consistent with this view, genome-wide maps of Spo11-DSB formation generated in the absence of Tel1 are skewed towards regions that load pro-DSB factors early--revealing regions of preferential priming--but this effect is abolished when NDT80 is deleted. Our work highlights how the stochastic nature of Spo11-DSB formation in individual cells within the limited temporal window of meiotic prophase can cause localised DSB clustering--a phenomenon that is exacerbated in tel1{Delta} cells due to the dual roles that Tel1 has in DSB interference and meiotic prophase checkpoint control.

molecular biology↗

bioRxiv2019

Concerted cutting by Spo11 illuminates DNA break mechanisms and initiates gap repair during meiosis

Genetic variation arises during meiosis via repair of DNA double-strand breaks (DSBs) created by the topoisomerase-like Spo11 protein. These DSBs are thought to always occur sparsely across the genome, with isolated DSBs generating discrete recombination events. We challenge this view, demonstrating that hyper-localised coincident DSBs frequently form within hotspots in both S. cerevisiae and mouse--a process suppressed by the DNA damage response kinase Tel1/ATM. Remarkably, the distances separating coincident DSBs vary with [~]10.5 bp periodicity, invoking a model where adjacent Spo11 molecules have a fixed orientation relative to the DNA helix. Deep sequencing of meiotic progeny identifies recombination scars consistent with gap repair initiated by adjacent DSBs. Our results revise current thinking about how genetic recombination initiates, reviving original concepts of meiotic recombination as double-strand gap repair.

molecular biology↗

bioRxiv2019

Convergent genes shape budding yeast pericentromeres

The 3D architecture of the genome governs its maintenance, expression and transmission. The conserved ring-shaped cohesin complex organises the genome by topologically linking distant loci on either a single DNA molecule or, after DNA replication, on separate sister chromatids to provide the cohesion that resists the pulling forces of spindle microtubules during mitosis1,2. Cohesin is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres3-7. However, the structural organisation of pericentromeres and implications for chromosome segregation are unknown. Here we report the 3D structure of budding yeast pericentromeres and establish the relationship between genome organisation and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organised into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Re-orienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation in mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading at centromeres shapes pericentromeres into a structure competent for chromosome segregation during mitosis. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded and the re-structuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between 3D genome organization of a specific chromosomal domain and cellular function.

cell biology↗

bioRxiv2019

A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes--and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy enables us to reveal the influence primary DNA sequence has upon Top2 cleavage--distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

molecular biology↗

bioRxiv2018

Separable roles of the DNA damage response kinase Mec1(ATR) and its activator Rad24(RAD17) within the regulation of meiotic recombination

During meiosis, programmed DNA double-strand breaks (DSBs) are formed by the topoisomerase-like enzyme, Spo11, activating the DNA damage response (DDR) kinase Mec1ATR via the checkpoint clamp loader, Rad24RAD17. At single loci, loss of Mec1 and Rad24 activity alters Spo11-dependent DSB formation and recombination outcome, but their genome-wide roles have not been examined in detail. Here we utilise two distinct methods to characterise the roles Mec1 and Rad24 play in meiotic recombination--deletion of the mismatch repair protein, Msh2, and control of meiotic prophase length via regulation of the Ndt80 transcription factor--to enable genome-wide mapping of meiotic progeny. In line with previous studies, we observe a reduction in the frequency of recombination upon deletion of RAD24--driven by a shortened prophase. By contrast, loss of Mec1 function increases recombination frequency, consistent with its role in DSB trans-interference. Despite this difference in recombination rate, complex, multi-chromatid events initiated by closely spaced DSBs--a rare event in wild type cells--occur more frequently in the absence of either Rad24 or Mec1, suggesting a loss of spatial regulation at the level of DSB formation. We further demonstrate that Mec1 and Rad24 also have important, yet distinct, roles in the spatial regulation of crossovers (COs). Specifically, excess DSBs forming in the absence of Mec1 are disproportionately channelled into the non-crossover (NCO) and non-interfering CO pathways, reducing the global strength of interference without an explicit change in the frequency of interfering COs. In direct contrast, loss of Rad24 weakens interference via a reduction in the apparent number of interfering COs--similar, but less extreme, to the phenotype of ZMM mutants such as zip3{Delta}. Collectively, our results highlight novel and unique roles for Rad24 within meiotic recombination--beyond those mediated by activation of Mec1--and describe new roles for the DDR in several important aspects of meiosis.

genetics↗

bioRxiv2018

Mismatch repair impedes meiotic crossover interference

Sequence divergence, mediated by the anti-recombinogenic activity of mismatch repair (MMR), forms a barrier to meiotic recombination and in turn the formation of viable gametes. However, rather than MMR acting as a non-specific impediment to meiotic recombination, here we provide evidence that at regions of greater sequence divergence MMR preferentially suppresses interfering (class I) crossovers (COs). Specifically, as measured in two Saccharomyces cerevisiae hybrids containing thousands of DNA-sequence polymorphisms, removal of MMR components increases both the frequency of CO formation and the uniformity of the observed CO distribution. At fine scale, CO positions are skewed away from polymorphic regions in MMR-proficient cells, but, critically, not when members of the class I CO pathway, MSH4 or ZIP3, are inactivated. These findings suggest that class I COs are more sensitive to heteroduplex DNA arising during recombination. Simulations and analysis of Zip3 foci on meiotic chromosomes support roles for Msh2 both early and late in the class I CO maturation process. Collectively, our observations highlight an unexpected interaction between DNA sequence divergence, MMR, and meiotic class I CO control, thereby intimately linking the regulation of CO numbers and their distribution to pathways contributing to reproductive isolation and eventual speciation.

genetics↗

bioRxiv2018

Principles of Meiotic Chromosome Assembly

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we elucidate how this elaborate three-dimensional chromosome organisation is underpinned by genomic sequence in Saccharomyces cerevisiae. Entering meiosis, strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion limited by barriers, yet are patterned by convergent transcription rather than binding of the mammalian interphase factor, CTCF, which is absent in S. cerevisiae--thereby both challenging and extending current paradigms of local chromosome organisation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process.

molecular biology↗