Mus81 nuclease and Sgs1 helicase are essential for meiotic recombination in a protist lacking a synaptonemal complex

Lukaszewicz, Agnieszka; Howard-Till, Rachel A.; Loidl, Josef

doi:10.1093/nar/gkt703

Cited by 29 publications

(34 citation statements)

References 78 publications

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“…Recent studies (Plohl et al, 2014; Topp and Dawe, 2006) have greatly increased understanding of centromere structure and function, but much still remains unclear. Several biological features of Tetrahymena , as well as its powerful experimental toolbox, have made this organism a useful model for studies of centromeric heterochromatin (Cervantes et al, 2006; Cui and Gorovsky, 2006; Papazyan et al, 2014), recombination (Lukaszewicz et al, 2013; Shodhan et al, 2014), chromosome cohesion (Howard-Till et al, 2013), and centromere evolution (Elde et al, 2011; Malik and Henikoff, 2002, 2009), all of which would benefit from better genetic and molecular definition of its centromeres. The full-length chromosomal super-assemblies described above make this possible.…”

Section: Resultsmentioning

confidence: 99%

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

Hamilton

Kapusta

Huvos

et al. 2016

eLife

145

264

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The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena’s germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.DOI: http://dx.doi.org/10.7554/eLife.19090.001

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Section: Resultsmentioning

confidence: 99%

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

Hamilton

Kapusta

Huvos

et al. 2016

eLife

145

264

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“…In conclusion, factors directly involved in generating CO and NCO recombinants during meiosis have been identified and characterized in recent years [12][13][14][15]21 , and several inroads have been made in understanding how template choice is regulated and executed during meiotic recombination 10,28 . However, we still only have a basic understanding of how underlying DNA sequence polymorphisms influence meiotic recombination outcomes.…”

Section: Fml1 Is a Negative Modulator Of Co Frequency Among Gene Convmentioning

confidence: 99%

“…pombe) has been shown to limit CO formation in fission yeast and Arabidopsis 12,13 . RecQ-type DNA helicases perform a wide range of regulatory roles in homologous recombination, and one of which probably is the promotion of NCO formation during meiosis in various organisms [14][15][16][17][18] .…”

Section: Introductionmentioning

confidence: 99%

DNA sequence differences are determinants of meiotic recombination outcome

Brown

Asogwa

Jézéquel

et al. 2019

Preprint

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PreambleDue to a mistake during analysis of a batch of Sanger sequencing reactions for Supplementary Figure S1, we erroneously stated that we found evidence for intragenic crossovers. We now show that intragenic crossovers are less likely than we initially thought. We sincerely apologize for our mishap and any inconvenience it might have caused. However, this does not affect the main conclusions of our paper, just how some of our results are interpreted. This new manuscript version has been amended accordingly. AbstractMeiotic recombination is essential for producing healthy gametes, and also generates genetic diversity. DNA double-strand break (DSB) formation is the initiating step of meiotic recombination, producing, among other outcomes, crossovers between homologous chromosomes (homologs), which provide physical links to guide accurate chromosome segregation. The parameters influencing DSB position and repair are thus crucial determinants of reproductive success and genetic diversity. Using Schizosaccharomyces pombe, we show that the distance between sequence polymorphisms across homologs has a strong impact on meiotic recombination rate. The closer the sequence polymorphisms are to each other across the homologs the fewer recombination events were observed. In the immediate vicinity of DSBs, sequence polymorphisms affect the frequency of intragenic recombination events (gene conversions). Additionally, and unexpectedly, the crossover rate of flanking markers tens of kilobases away from the sequence polymorphisms was affected by their relative position to each other amongst the progeny having undergone intragenic recombination. A major regulator of this distance-dependent effect is the MutSα-MutLα complex consisting of Msh2, Msh6, Mlh1, and Pms1. Additionally, the DNA helicases Rqh1 and Fml1 shape recombination frequency, although the effects seen here are largely independent of the relative position of the sequence polymorphisms.

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“…In contrast to the gene KO strategies, RNAi KD can be done in a shorter period of time and is thus suitable for high-throughput genetic screening. However, RNAi KD often only incompletely shuts down expression of a target gene (Howard-Till and Yao 2006;Cheng et al 2010;Chung and Yao 2012;Lukaszewicz et al 2013) and may thus result in the misinterpretation of the gene function. During the development of the new MAC of Tetrahymena, .8000 DNA segments called internal eliminated sequences (IESs) were reproducibly eliminated.…”

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confidence: 99%

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

Hayashi¹,

Mochizuki

2015

Genetics

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Tetrahymena is a useful eukaryotic model for biochemistry and molecular cell biology studies. We previously demonstrated that targeted ectopic DNA elimination, also called co-Deletion (coDel), can be induced by the introduction of an internal eliminated sequence (IES)-target DNA chimeric construct. In this study, we demonstrate that coDel occurs at most of the loci tested and can be used for the production of somatic gene KO strains. We also showed that coDel at two loci can be simultaneously induced by a single transformation; thus, coDel can be used to disrupt multiple gene loci in a single cell. Therefore, coDel is a useful tool for functional genetics in Tetrahymena and further extends the usefulness of this model organism.KEYWORDS Tetrahymena; DNA elimination; RNAi; gene knockout T HE ciliated protozoan Tetrahymena thermophila can grow at an exceptionally high rate (its doubling time is approximately 2 hr) and can reach a high density (a few million cells per milliliter) under simple and inexpensive culture conditions (reviewed in Orias et al. 2000). In combination with robust genetic manipulation methods (reviewed in Chalker 2012), Tetrahymena is a useful model eukaryote for biochemistry and molecular cell biology studies (reviewed in Collins and Gorovsky 2005).Three strategies for loss-of-function genetic studies have been established for this organism. The first strategy is a germline knockout (KO), in which one of the two gene copies in the diploid micronucleus (MIC) is replaced with a drug-resistance gene by homologous recombination, and two heterozygous strains are then sexually crossed to obtain a homozygous germline KO strain (Cassidy-Hanley et al. 1997). The second strategy is somatic KO, in which one of the 45 copies of a gene in the polyploid macronucleus (MAC) is replaced with a drugresistance gene by homologous recombination, and "phenotypic assortment" is used to obtain cells with the drug-resistance gene at all loci (Merriam and Bruns 1988). Phenotypic assortment is possible because MAC chromosomes are randomly segregated into daughter cells in vegetative cell division and a stepwise increase of the drug in culture selects for cells that have more drug-resistance genes. The final strategy is gene knockdown (KD) by RNA interference (RNAi), in which a construct expressing long hairpin RNA that is complementary to the gene of interest is introduced into a nonessential locus in the MAC and small RNAs produced from the hairpin RNA posttranscriptionally silence the expression of the gene (HowardTill and Yao 2006).Although the first two gene KO strategies have been reliably used to study individual gene functions, they are not suitable for high-throughput genetic screening because the integration of a KO construct into the MIC germline occurs at very low efficiency and the phenotypic assortment process used in somatic KOs require the careful control of drug concentrations in each strain and culture step. Moreover, it takes 1 month to obtain KO strains using these methods. For germline KO, t...

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Mus81 nuclease and Sgs1 helicase are essential for meiotic recombination in a protist lacking a synaptonemal complex

Cited by 29 publications

References 78 publications

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

DNA sequence differences are determinants of meiotic recombination outcome

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

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