Sir2p suppresses recombination of replication forks stalled at the replication fork barrier of ribosomal DNA in Saccharomyces cerevisiae

6 p.-3 fig.In the ribosomal DNA (rDNA) of Saccharomyces cerevisiae replication forks progressing against transcription stall at a polar replication fork barrier (RFB) located close to and downstream of the 35S transcription unit. Forks blocked at this barrier are potentially recombinogenic. Plasmids bearing the RFB sequence in its active orientation integrated into the chromosomal rDNA in sir2 mutant cells but not in wild-type cells, indicating that the histone deacetylase silencing protein Sir2 (Sir2p), which also modulates the aging process in yeast, suppresses the recombination competence of forks blocked at the rDNA RFB. Orientation of the RFB sequence in its inactive course or its abolition by FOB1 deletion avoided plasmid integration in sir2 mutant cells, indicating that stalling of the forks in the plasmid context was required for recombination to take place. Altogether these results strongly suggest that one of the functions of Sir2p is to modulate access of the recombination machinery to the forks stalled at the rDNA RFB.This work was partially supported by grants PGC PB98-048 from the Spanish Comisión Interministerial de Ciencia y Tecnología, SAF2001-1740 from the Spanish Ministerio de Ciencia y Tecnología and 08.5/0057/2001.1 and 08.1/0067/2001.1 from the Comunidad Autónoma de Madrid.Peer reviewe

Plasmid DNA replication and topology as visualized by two-dimensional agarose gel electrophoresis

31 p.- 12 fig.During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, replication fork reversal, the interplay of supercoiling and catenation and other changes in DNA topology that take place as replication progresses.This work was sustained in part by Grants # BIO2005-02224 and BFU2008-00408/BMC to J.B.S. and BFU2007-62670 to P.H. from the Spanish Ministerio de Ciencia e Innovación.Peer reviewe

Interplay of DNA supercoiling and catenation during the segregation of sister duplexes

The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in viv

Changes in the Topology of DNA Replication Intermediates: In vivo vs In vitro

Most of the methods used to analyze DNA, including electrophoresis, electron microscopy or atomic force microscopy, involve de-proteinization, and it is well known that the removal of proteins affects DNA topology. After de-proteinization in vitro, the topology of replication intermediates changes significantly. A comprehensive analysis of the topological changes introduced during DNA isolation (de-proteinization) is important to get a better understanding of DNA topology in vivo. The topology of replication intermediates examined by electrophoresis, electron microscopy or atomic force microscopy in vitro does not necessarily represent the situation in vivo.CONACYT - Consejo Nacional de Ciencias y TecnologíaPROCIENCI

Dynamics of torsionally stressed DNA replication intermediates

As an extension of this study we project the simulation of DNA molecules with a size similar to that of DNA circles that are capable to self-replicate. We also want to expand the study to other mechanical and thermodynamic properties of replication intermediates..CONACYT - Consejo Nacional de Ciencias y TecnologíaPROCIENCI

Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates

DNA topoisomerases are the enzymes that regulate DNA topology in all living cells. Since the discovery and purification of ω (omega), when the first were topoisomerase identified, the function of many topoisomerases has been examined. However, their ability to relax supercoiling and unlink the pre-catenanes of partially replicated molecules has received little attention. Here, we used two-dimensional agarose gel electrophoresis to test the function of three type II DNA topoisomerases in vitro: the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α. We examined the proficiency of these topoisomerases on a partially replicated bacterial plasmid: pBR-TerE@AatII, with an unidirectional replicating fork, stalled when approximately half of the plasmid had been replicated in vivo. DNA was isolated from two strains of Escherichia coli: DH5αF’ and parE10. These experiments allowed us to assess, for the first time, the efficiency of the topoisomerases examined to resolve supercoiling and pre-catenanes in partially replicated molecules and fully replicated catenanes formed in vivo. The results obtained revealed the preferential functions and also some redundancy in the abilities of these DNA topoisomerases in vitro

Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresi

Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication

DNA topology plays a crucial role in all living cells. In prokaryotes, negative supercoiling is required to initiate replication and either negative or positive supercoiling assists decatenation. The role of DNA knots, however, remains a mystery. Knots are very harmful for cells if not removed efficiently, but DNA molecules become knotted in vivo. If knots are deleterious, why then does DNA become knotted? Here, we used classical genetics, high-resolution 2D agarose gel electrophoresis and atomic force microscopy to show that topoisomerase IV (Topo IV), one of the two type-II DNA topoisomerases in bacteria, is responsible for the knotting and unknotting of sister duplexes during DNA replication. We propose that when progression of the replication forks is impaired, sister duplexes become loosely intertwined. Under these conditions, Topo IV inadvertently makes the strand passages that lead to the formation of knots and removes them later on to allow their correct segregation

Presence of polysaccharides and proteins in the chromatoid body of mouse spermatids

6 p.- 8 fig.We have carried out a cytochemical analysis of the chromatoid body employing preferential methods for the detection of basic proteins and polysaccharides. The chromatoid body appears possitive after alcoholic PTA staining suggesting a basic protein composition.Vesicles surrounding the chromatoid body appear positive after aqueous PTA and the Tlhiery procedure. The presence of polysaccharides in the vesicles of the chromatoid body,and a re1ationship be-tween them and the Colgi complex is suggested.Peer reviewe

Preferential staining of the post-acrosomal lamina of mouse spermatids

5 p.-4 fig.Testes from adult mouse, after silver impregnation, were analyzed under the light and electron microscopes. In young spermatids the nucleolus shows a strong contrast and some silver granules appear in the nucleus. In all cases the cytplasmic structures appear devoid of silver granules. The post-acrosomal lamina un adult spermatids shows preferential staining. The importance of the selective staining of this lamina is discussed in relation to the physiology of the sperm.Peer reviewe