Parameters in gene conversion: An algebraic analysis of the hybrid DNA model at the gray locus of Sordaria fimicola

We have extended previous algebraic analyses of aberrant segregation at the gray locus of Sordaria fimicola (Whitehouse, 1965; Emerson, 1966; Fincham, Hill & Reeve, 1980) to the more complex situation where aberrant segregations are detected in three factor crosses involving two flanking markers. This algebra has been applied to seven gray alleles which have been extensively characterized for their pattern of gene conversion and postmeiotic segregation by Kitani & Olive (1967). It is based on seven major types of aberrant segregation which can be distinguished in the presence of flanking markers spanning the converting site, and allows us to use up to six parameters to describe hDNA formation and mismatch repair. We present solutions which predict a spectrum of aberrant segregation fitting the experimental data at the P > 0·05 level for six of the seven alleles tested. They are consistent with the following properties of hDNA at the gray locus: (1) the single stranded DNA transferred during hDNA formation has always the same chemical polarity. (2) hDNA is mostly, if not entirely, symmetric, and its probability of formation is constant over the whole gene. (3) Disparity in aberrant segregation is mostly, if not entirely due to disparity in mismatch repai

Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III

RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner

Non-canonical DNA transcription enzymes and the conservation of two-barrel RNA polymerases

DNA transcription depends on multimeric RNA polymerases that are exceptionally conserved in all cellular organisms, with an active site region of >500 amino acids mainly harboured by their Rpb1 and Rpb2 subunits. Together with the distantly related eukaryotic RNA-dependent polymerases involved in gene silencing, they form a monophyletic family of ribonucleotide polymerases with a similarly organized active site region based on two double-Ψ barrels. Recent viral and phage genome sequencing have added a surprising variety of putative nucleotide polymerases to this protein family. These proteins have highly divergent subunit composition and amino acid sequences, but always contain eight invariant amino acids forming a universally conserved catalytic site shared by all members of the two-barrel protein family. Moreover, the highly conserved ‘funnel’ and ‘switch 2’ components of the active site region are shared by all putative DNA-dependent RNA polymerases and may thus determine their capacity to transcribe double-stranded DNA templates

Mutations of RNA polymerase II activate key genes of the nucleoside triphosphate biosynthetic pathways

The yeast URA2 gene, encoding the rate-limiting enzyme of UTP biosynthesis, is transcriptionally activated by UTP shortage. In contrast to other genes of the UTP pathway, this activation is not governed by the Ppr1 activator. Moreover, it is not due to an increased recruitment of RNA polymerase II at the URA2 promoter, but to its much more effective progression beyond the URA2 mRNA start site(s). Regulatory mutants constitutively expressing URA2 resulted from cis-acting deletions upstream of the transcription initiator region, or from amino-acid replacements altering the RNA polymerase II Switch 1 loop domain, such as rpb1-L1397S. These two mutation classes allowed RNA polymerase to progress downstream of the URA2 mRNA start site(s). rpb1-L1397S had similar effects on IMD2 (IMP dehydrogenase) and URA8 (CTP synthase), and thus specifically activated the rate-limiting steps of UTP, GTP and CTP biosynthesis. These data suggest that the Switch 1 loop of RNA polymerase II, located at the downstream end of the transcription bubble, may operate as a specific sensor of the nucleoside triphosphates available for transcription

Parameters in gene conversion: An algebraic analysis of the hybrid DNA model at the gray locus of Sordaria fimicola.

We have extended previous algebraic analyses of aberrant segregation at the gray locus of Sordaria fimicola (Whitehouse, 1965; Emerson, 1966; Fincham, Hill & Reeve, 1980) to the more complex situation where aberrant segregations are detected in three factor crosses involving two flanking markers. This algebra has been applied to seven gray alleles which have been extensively characterized for their pattern of gene conversion and postmeiotic segregation by Kitani & Olive (1967). It is based on seven major types of aberrant segregation which can be distinguished in the presence of flanking markers spanning the converting site, and allows us to use up to six parameters to describe hDNA formation and mismatch repair. We present solutions which predict a spectrum of aberrant segregation fitting the experimental data at the P > 0·05 level for six of the seven alleles tested. They are consistent with the following properties of hDNA at the gray locus: (1) the single stranded DNA transferred during hDNA formation has always the same chemical polarity. (2) hDNA is mostly, if not entirely, symmetric, and its probability of formation is constant over the whole gene. (3) Disparity in aberrant segregation is mostly, if not entirely due to disparity in mismatch repair

Existence de gènes régulateurs couplant la répression de la biosynthèse et l'induction du catabolisme de l'arginine dans Saccharomyces cerevisiae

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Domaines fonctionnels de l'ARN polymérase II chez Saccharomyces cerevisiae (transcription, clivage du transcrit et couplage à la réparation)

Ce travail porte sur l'ARN polymérase II de la levure Saccharomyces cerevisiæ. Sa première partie expérimentale concerne l'organisation du site actif des ARN polymérases de levure, au niveau de trois domaines extrêmement conservés, le motif GY..ED de la sous-unité Rpb2, associé à l'un des deux Mg++ catalytiques (MgB), la boucle basique (GDK...R.GQKG) de la même sous-unité, liant le bout 3'OH de l'ARN et la boucle acide Q.RSADE du facteur d' élongation TFIIS. Ce travail fait l'objet d'un article, actuellement en révision à Nucleic Acids Research. J'ai ensuite caractérisé des mutants de la boucle 1 (switch loop 1) étudiés dans le cadre d'une collaboration avec François Lacroute. Dans la dernière partie de ce travail, j'ai évalué la sensibilité des mutants d'ARN polymérases aux UV, afin de mieux comprendre la réparation couplée à la transcription (TCR), un mécanisme favorisant la réparation de l'ADN au niveau des brins transcrits. Ces données renforcent l'hypothèse d'une contribution directe de l'ARN polymérase II à ce mode de réparation. Elles indiquent en outre que l'ATPase ADN-dépendante Rad26, orthologue de la protéine humaine CSB, ne contribue pas à la croissance cellulaire, elle est donc seulement requise pour la réparation couplée à la transcription.DNA-dependent RNA polymerases transcribe DNA, have a RNA cleavage activity and interact with DNA repair by facilitating the removal of cylobutane dimers on the transcribed DNA strand. Their active site is formed of several highly conserved motifs organised around two catalytic Mg++. In the first part of this study, I investigated three of these motifs : the acidic loop (GY..ED) of subunit Rpb2, thought to bind Mg(B), the basic loop (GDK...R.GQKG) of Rpb2, holding the RNA 3'-OH end, and the acidic loop (Q.RSADE) of the TFIIS elongation factor. I also contributed to the genetic study of a third loop, Switch 1, conserved in all non-bacterial RNA polymerases and possibly mediating a transcriptional response to nucleotide triphosphate shortage. Finally, a characterisation of the UV-response in RNA polymerase II mutants supported the hypothesis that RNA polymerase directly contribute to the fast repair of the transcribed strand of DNA.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Les ARN polymérases chez Saccharomyces cerevisiae (étude des sous-unités Rpb5, Rpb7 et Rpc25)

Chez les eucaryotes, comme Saccharomyces cerevisiae, la transcription de l'ADN est assurée par trois enzymes: l'ARN polymérase I, l'ARN polymérase II et l'ARN polymérase III, qui synthétisent respectivement les ARN ribosomiques, les ARN messagers et les ARN de transfert. On sait aujourd'hui qu'elles ont une structure très conservée, faite de onze sous-unités dont cinq correspondent aux sous-unités de l'enzyme bactérienne, et six sont partagées avec les archées. La douzième sous-unité est typiquement eucaryote. Dans ce travail, nous traitons de deux composantes partagées avec les archées, la sous-unité commune, Rpb5 et la sous-unité Rpc25 de l'ARN polymérase III. Nous avons montré que les mutants de Rpb5 affectent les trois ARN polymérases ce qui conforte l'idée de son rôle commun dans les trois systèmes. Par ailleurs cette fonction semble conservée au cours de l'évolution, puisque des protéines chimères composées de plus de 90 % de séquence humaine sont fonctionnelles dans la levure. De plus, nous avons observé une colétalité entre des mutants de Rpb5 et les mutants nuls rpb9-delta et rpb4-delta de l'ARN Pol II. Ainsi, Rpb9 et Rpb4 pourraient coopérer avec Rpb5 pour stabiliser l'ARN polymérase II et structurer le site actif de l'enzyme. Nous avons établi que la sous-unité Rpc25 de l'ARN Pol III se lie à la sous-unité Rpc17 et forme un hétérodimère conservé au cours de l'évolution puisque Rpc17 et Rpc25 sont, respectivement, les orthologues des sous-unités RpoF et RpoE de l'ARN polymérase archébactérienne. Ce complexe se retrouve aussi dans les ARN polymérases I et II : Rpa14/Rpa43 et Rpb4/Rpb7, respectivement. De plus, nous avons établi que des mutants de Rpc25 sont affectés, in vitro, dans l'initiation de la transcription, et notamment dans la reconnaissance du site de pré-initiation. Ainsi, dans l'ARN Pol III l'hétérodimère Rpc17/Rpc25 jouerait un rôle dans l'initiation de la transcription, comme c'est le cas pour les complexes paralogues dans les ARN Pol I et ARN Pol II.In eukaryotes, like Saccharomyces cerevisiae, RNA is transcribed by three enzymes: RNA polymerases I, II and III that synthesize respectively ribosomal RNAs, messenger RNAs and transfer RNAs. It has been established that they have a conserved structure made of eleven subunits; five of them correspond to bacterial enzyme and six are conserved with archaea. The twelfth is solely eukaryote. This work deals with two components shared with archaea: the common subunit Rpb5 and a subunit of RNA Pol III, Rpc25. We showed here that mutants of Rpb5 affect the three RNA polymerases, in vivo, supporting the idea of a common role in all three transcriptional systems. Moreover, its function seems to be conserved, since hybrid proteins with up 90 % of the human sequence are functional in yeast. We observed a synthetic lethality between mutants of Rpb5 and null mutants rpb4-delta and rpb9-delta, in RNA Pol II. Thus, Rpb4 and Rpb9 may cooperate with Rpb5 to stabilise and structure the active site of RNA Pol-II. We established that the subunit Rpc25 of RNA Pol III binds to Rpc 17 and forms an heterodimer, conserved through evolution. Indeed, Rpc17 and Rpc25 are, respectively, the orthologues of RpoF and RpoE in archaea. This complex is also found in RNA PolI and RNA Pol II: Rpa14/Rpa43 and Rpb4/Rpb7, respectively. We also demonstrated that rpc25 mutants are impaired in the initiation step of RNA Pol III transcription, particularly in the recognition of the pre-initiation complex.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The IFH1 gene product interacts with a fork head protein in Saccharomyces cerevisiae

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