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

RNA-SEQ of fv-induced erythroleukemia cells suggests a role of wasp.

The goal of research is analyze the molecular function of was in progenitors of the erythoid linage and its role in reprogramming celular differentiation in erythroleukemia cells.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

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Supercoiling drives DNA unknotting and postreplicative decatenation

El superenrollamiento impulsa el desanudamiento del ADN y la decatenación posreplicativa.CONACYT – Consejo Nacional de Ciencia y Tecnologí

Analysis of DNA topology of EBV minichromosomes in HEK 293 cells

16 p.-8 fig.Simian Virus 40 (SV40) and Epstein-Barr Virus (EBV) are frequently used as model systems to study DNA replication. Their genomes are both circular duplex DNAs organized in a single replicon where replication initiates at a precise site upon binding of a specific protein: the large tumor (T) antigen for SV40 and the Epstein-Barr Nuclear Antigen 1 (EBNA-1) for EBV. Despite the abundant information available on the genetics and biochemistry of the replication process in these systems, little is known about the changes in DNA topology that take place as molecules are transfected into eukaryotic cells, assembled into chromatin and bind initiator proteins to start replication. Here we used high-resolution two-dimensional agarose gel electrophoresis to demonstrate that in Human Embryonic Kidney (HEK) 293 cells, minichromosomes of almost the same mass carrying either the SV40 or the EBV replication origin showed similar topological features. The patterns were very similar regardless of the initiator proteins. We also showed that in a hybrid minichromosome, pEco3’Δ, that initiates replication from the SV40 origin, the presence of EBNA-1 and its putative binding to the EBV “family of repeats” induces no significant topological change. These observations challenge the idea that binding of EBNA-1 to oriP could induce negative supercoiling and favor a model suggesting that it binds to oriP in a two-step process where only the second step causes structural changes in a transient cell cycle specific manner.This work was supported by Grant 14-INV-062 from the Paraguayan CONACYT-PROCIENCIA program to MJFN; Grant BFU2014-56835 from the Spanish Ministerio de Economía y Competitividad to JBS.Peer reviewe

PU.1 is dispensable to block erythroid differentiation in Friend erythroleukemia cells

10 páginas, 7 figuras -- PAGS nros. 121-130Friend murine erythroleukemia cell lines derive from erythroblasts transformed with the Friend complex where the spleen-focus forming virus integrated in the vicinity of the Sfpi-1 locus. Erythroleukemia cells do not differentiate and grow indefinitely in the absence of erythropoietin. Activation of the transcription factor PU.1, encoded by the Sfpi-1 gene, is thought to be responsible for the transformed phenotype. These cells can overcome the blockage and reinitiate their differentiation program when exposed to some chemical inducers such as hexamethylene bisacetamide. In this study, we established cell cultures that were capable to proliferate unconstrained in the presence of the inducer. Resistant cell lines restart erythroid differentiation, though, if forced to exit the cell cycle or by overexpressing the transcription factor GATA-1. Unexpectedly, expression of PU.1 was suppressed in the resistant clones albeit the spleen-focus forming virus was still integrated in the proximity of the Sfpi-1 locus. Exposure to 5-Aza-2′-deoxycytidine activates PU.1 expression suggesting that the PU.1 coding gene is highly methylated in the resistant cells. Altogether these results suggest that PU.1 is dispensable to block erythroid differentiationThis work was supported in part by the Spanish Ministerio de Educación y Ciencia Grants BIO2005-02224 and BFU2004-00125. MJFN is supported by the Consejería de Educación de la Comunidad de Madrid, Fondo Social Europeo and a fellowship from the Residencia de Estudiantes (CSIC)-Ayuntamiento de MadridPeer reviewe

PU.1 is dispensable to block erythroid differentiation in Friend erythroleukemia cells

10 páginas, 7 figuras -- PAGS nros. 121-130Friend murine erythroleukemia cell lines derive from erythroblasts transformed with the Friend complex where the spleen-focus forming virus integrated in the vicinity of the Sfpi-1 locus. Erythroleukemia cells do not differentiate and grow indefinitely in the absence of erythropoietin. Activation of the transcription factor PU.1, encoded by the Sfpi-1 gene, is thought to be responsible for the transformed phenotype. These cells can overcome the blockage and reinitiate their differentiation program when exposed to some chemical inducers such as hexamethylene bisacetamide. In this study, we established cell cultures that were capable to proliferate unconstrained in the presence of the inducer. Resistant cell lines restart erythroid differentiation, though, if forced to exit the cell cycle or by overexpressing the transcription factor GATA-1. Unexpectedly, expression of PU.1 was suppressed in the resistant clones albeit the spleen-focus forming virus was still integrated in the proximity of the Sfpi-1 locus. Exposure to 5-Aza-2′-deoxycytidine activates PU.1 expression suggesting that the PU.1 coding gene is highly methylated in the resistant cells. Altogether these results suggest that PU.1 is dispensable to block erythroid differentiationThis work was supported in part by the Spanish Ministerio de Educación y Ciencia Grants BIO2005-02224 and BFU2004-00125. MJFN is supported by the Consejería de Educación de la Comunidad de Madrid, Fondo Social Europeo and a fellowship from the Residencia de Estudiantes (CSIC)-Ayuntamiento de MadridPeer reviewe