Person: Kleckner, Nancy
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Kleckner
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Nancy
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Kleckner, Nancy
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Publication The challenge of evolving stable polyploidy: could an increase in “crossover interference distance” play a central role?(Springer Berlin Heidelberg, 2016) Bomblies, Kirsten; Jones, Gareth; Franklin, Chris; Zickler, Denise; Kleckner, NancyWhole genome duplication is a prominent feature of many highly evolved organisms, especially plants. When duplications occur within species, they yield genomes comprising multiple identical or very similar copies of each chromosome (“autopolyploids”). Such genomes face special challenges during meiosis, the specialized cellular program that underlies gamete formation for sexual reproduction. Comparisons between newly formed (neo)-autotetraploids and fully evolved autotetraploids suggest that these challenges are solved by specific restrictions on the positions of crossover recombination events and, thus, the positions of chiasmata, which govern the segregation of homologs at the first meiotic division. We propose that a critical feature in the evolution of these more effective chiasma patterns is an increase in the effective distance of meiotic crossover interference, which plays a central role in crossover positioning. We discuss the findings in several organisms, including the recent identification of relevant genes in Arabidopsis arenosa, that support this hypothesis. Electronic supplementary material The online version of this article (doi:10.1007/s00412-015-0571-4) contains supplementary material, which is available to authorized users.Publication Dynamic Trans Interactions in Yeast Chromosomes(Public Library of Science, 2013) Mirkin, Ekaterina V.; Chang, Frederick S.; Kleckner, NancyThree-dimensional organization of the genome is important for regulation of gene expression and maintenance of genomic stability. It also defines, and is defined by, contacts between different chromosomal loci. Interactions between loci positioned on different chromosomes, i.e. “trans” interactions are one type of such contacts. Here, we describe a case of inducible trans interaction in chromosomes of the budding yeast S. cerevisiae. Special DNA sequences, inserted in two ectopic chromosomal loci positioned in trans, pair with one another in an inducible manner. The spatial proximity diagnostic of pairing is observable by both chromosome capture analysis (3C) and epifluorescence microscopy in whole cells. Protein synthesis de novo appears to be required for this process. The three-dimensional organization of the yeast nucleus imposes a constraint on such pairing, presumably by dictating the probability with which the two sequences collide with one another.Publication Crossover Patterning by the Beam-Film Model: Analysis and Implications(Public Library of Science, 2014) Zhang, Liangran; Liang, Zhangyi; Hutchinson, John; Kleckner, NancyCrossing-over is a central feature of meiosis. Meiotic crossover (CO) sites are spatially patterned along chromosomes. CO-designation at one position disfavors subsequent CO-designation(s) nearby, as described by the classical phenomenon of CO interference. If multiple designations occur, COs tend to be evenly spaced. We have previously proposed a mechanical model by which CO patterning could occur. The central feature of a mechanical mechanism is that communication along the chromosomes, as required for CO interference, can occur by redistribution of mechanical stress. Here we further explore the nature of the beam-film model, its ability to quantitatively explain CO patterns in detail in several organisms, and its implications for three important patterning-related phenomena: CO homeostasis, the fact that the level of zero-CO bivalents can be low (the “obligatory CO”), and the occurrence of non-interfering COs. Relationships to other models are discussed.Publication The differential extension in dsDNA bound to Rad51 filaments may play important roles in homology recognition and strand exchange(Oxford University Press, 2013) Danilowicz, Claudia; Peacock-Villada, Alexandra; Vlassakis, Julea; Facon, Adrien; Feinstein, Efraim; Kleckner, Nancy; Prentiss, MaraRecA and Rad51 proteins play an important role in DNA repair and homologous recombination. For RecA, X-ray structure information and single molecule force experiments have indicated that the differential extension between the complementary strand and its Watson–Crick pairing partners promotes the rapid unbinding of non-homologous dsDNA and drives strand exchange forward for homologous dsDNA. In this work we find that both effects are also present in Rad51 protein. In particular, pulling on the opposite termini (3′ and 5′) of one of the two DNA strands in a dsDNA molecule allows dsDNA to extend along non-homologous Rad51-ssDNA filaments and remain stably bound in the extended state, but pulling on the 3′5′ ends of the complementary strand reduces the strand-exchange rate for homologous filaments. Thus, the results suggest that differential extension is also present in dsDNA bound to Rad51. The differential extension promotes rapid recognition by driving the swift unbinding of dsDNA from non-homologous Rad51-ssDNA filaments, while at the same time, reducing base pair tension due to the transfer of the Watson–Crick pairing of the complementary strand bases from the highly extended outgoing strand to the slightly less extended incoming strand, which drives strand exchange forward.Publication Double Holliday junctions are intermediates of DNA break repair(Nature Publishing Group, 2010) Bzymek, Malgorzata; Thayer, Nathaniel; Oh, Steve; Hunter, Neil; Kleckner, NancyRepair of DNA double-strand breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumour suppression. However, despite its importance, the molecular intermediates of mitotic DSB repair remain undefined. The double Holliday junc- tion (DHJ), presupposed to be the central intermediate for more than 25 years\(^1\), has only been identified during meiotic recombination\(^2\). Moreover, evidence has accumulated for alternative, DHJ-independent mechanisms\(^{3–6}\), raising the possibility that DHJs are not formed during DSB repair in mitotically cycling cells. Here we identify intermediates of DSB repair by using a budding-yeast assay system designed to mimic physiological DSB repair. This system uses diploid cells and provides the possibility for allelic recombination either between sister chromatids or between homologues, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homologue joint molecule (JM) intermediates whose strand composition and size are identical to those of the canonical DHJ structures observed in meiosis\(^2\). However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologues. Moreover, JMs seem to represent a minor pathway of DSB repair in mitotic cells, being detected at about tenfold lower levels (per DSB) than during meiotic recombination. Thus, although DHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.Publication Direct recognition of homology between double helices of DNA in Neurospora crassa(2014) Gladyshev, Eugene; Kleckner, NancyChromosomal regions of identical or nearly identical DNA sequence can preferentially associate with one another in the apparent absence of DNA breakage. Molecular mechanism(s) underlying such homology-dependent pairing phenomena remain(s) unknown. Using Neurospora crassa repeat-induced point mutation (RIP) as a model system, we show that a pair of DNA segments can be recognized as homologous if they share triplets of base pairs arrayed with the matching periodicity of 11 or 12 base pairs. This pattern suggests direct interactions between slightly underwound co-aligned DNA duplexes engaging once per turn and over many consecutive turns. The process occurs in the absence of MEI3, the only RAD51/DMC1 protein in N. crassa, demonstrating independence from the canonical homology recognition pathway. A new perspective is thus provided for further analysis of the breakage-independent recognition of homology that underlies RIP and, potentially, other processes where sequence-specific pairing of intact chromosomes is involved.Publication Interference-mediated synaptonemal complex formation with embedded crossover designation(Proceedings of the National Academy of Sciences, 2014) Zhang, Liangran; Espagne, Eric; de Muyt, Arnaud; Zickler, Denise; Kleckner, NancyBiological systems exhibit complex patterns, at length scales ranging from the molecular to the organismic. Along chromosomes, events often occur stochastically at different positions in different nuclei but nonetheless tend to be relatively evenly spaced. Examples include replication origin firings, formation of chromatin loops along chromosome axes and, during meiosis, designation of crossover recombination sites ("crossover interference"). We present evidence, in the fungus Sordaria macrospora, that crossover interference is part of a broader patterning program that includes synaptonemal complex (SC) nucleation. This program yields relatively evenly-spaced SC nucleation sites; among these, a subset is also crossover sites that show a classical interference distribution. This pattern ensures that SC forms regularly along the entire lengths of the chromosomes as required for homolog pairing maintenance and interlock sensing while concomitantly embedding crossover interactions within the SC structure as required for both DNA recombination and structural events of chiasma-formation. This pattern can be explained by a threshold-based interference process. This model can be generalized to give diverse types of related and/or partially overlapping patterns, in two or more dimensions, for any type of object.Publication Meiotic double-strand breaks occur once per pair of (sister) chromatids and, via Mec1/ATR and Tel1/ATM, once per quartet of chromatids(Proceedings of the National Academy of Sciences, 2011) Zhang, Liangran; Kim, Keun Pill; Kleckner, Nancy; Storlazzi, AuroraMeiotic recombination initiates via programmed double-strand breaks (DSBs). We investigate whether, at a given initiation site, DSBs occur independently among the four available chromatids. For a single DSB “hot spot”, the proportions of nuclei exhibiting zero, one, or two (or more) observable events were defined by tetrad analysis and compared with those predicted by different DSB distribution scenarios. Wild-type patterns are incompatible with independent distribution of DSBs among the four chromatids. In most or all nuclei, DSBs occur one-per-pair of chromatids, presumptively sisters. In many nuclei, only one DSB occurs per four chromatids, confirming the existence of trans inhibition where a DSB on one chromosome interactively inhibits DSB formation on the partner chromosome. Several mutants exhibit only a one-per-pair constraint, a phenotype we propose to imply loss of trans inhibition. Signal transduction kinases Mec1 (ATR) and Tel1 (ATM) exhibit this phenotype and thus could be mediators of this effect. Spreading trans inhibition can explain even spacing of total recombinational interactions and implies that establishment of interhomolog interactions and DSB formation are homeostatic processes. The two types of constraints on DSB formation provide two different safeguards against recombination failure during meiosis.Publication Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence(Public Library of Science, 2016) Gladyshev, Eugene; Kleckner, NancyHaploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. Once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. In Neurospora crassa this process is known as Repeat-Induced Point mutation (RIP). Previously, we showed that RIP did not require MEI-3, the only RecA homolog in Neurospora, and that it could detect homologous trinucleotides interspersed with a matching periodicity of 11 or 12 base-pairs along participating chromosomal segments. This pattern was consistent with a mechanism of homology recognition that involved direct interactions between co-aligned double-stranded (ds) DNA molecules, where sequence-specific dsDNA/dsDNA contacts could be established using no more than one triplet per turn. In the present study we have further explored the DNA sequence requirements for RIP. In our previous work, interspersed homologies were always examined in the context of a relatively long adjoining region of perfect homology. Using a new repeat system lacking this strong interaction, we now show that interspersed homologies with overall sequence identity of only 36% can be efficiently detected by RIP in the absence of any perfect homology. Furthermore, in this new system, where the total amount of homology is near the critical threshold required for RIP, the nucleotide composition of participating DNA molecules is identified as an important factor. Our results specifically pinpoint the triplet 5'-GAC-3' as a particularly efficient unit of homology recognition. Finally, we present experimental evidence that the process of homology sensing can be uncoupled from the downstream mutation. Taken together, our results advance the notion that sequence information can be compared directly between double-stranded DNA molecules during RIP and, potentially, in other processes where homologous pairing of intact DNA molecules is observed.Publication DNA sequence homology induces cytosine-to-thymine mutation by a heterochromatin-related pathway in Neurospora(2017) Gladyshev, Eugene; Kleckner, NancyEukaryotic genomes contain substantial amounts of repetitive DNA organized in the form of constitutive heterochromatin and associated with repressive epigenetic modifications, such as H3K9me3 and C5-cytosine methylation (5mC). In the fungus Neurospora crassa, H3K9me3 and 5mC are catalyzed, respectively, by a conserved SUV39 histone methyltransferase DIM-5 and a DNMT1-like cytosine methyltransferase DIM-2. Here we show that DIM-2 can also mediate Repeat-Induced Point mutation (RIP) of repetitive DNA in N. crassa. We further show that DIM-2-dependent RIP requires DIM-5, HP1, and other known heterochromatin factors, implying the role of a repeat-induced heterochromatin-related process. Our previous findings suggest that the mechanism of repeat recognition for RIP involves direct interactions between homologous double-stranded (ds) DNA segments. We thus now propose that, in somatic cells, homologous dsDNA/dsDNA interactions between a small number of repeat copies can nucleate a transient heterochromatic state, which, on longer repeat arrays, may lead to the formation of constitutive heterochromatin.