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

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

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

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

DNA knotting caused by head-on collision of transcription and replication

26 p.-4 fig.Collision of transcription and replication is uncommon, but the reason for nature to avoid this type of collision is still poorly understood. In Escherichia coli pBR322 is unstable and rapidly lost without selective pressure. Stability can be rescued if transcription of the tetracycline-resistance gene (TetR), progressing against replication, is avoided. We investigated the topological consequences of the collision of transcription and replication in pBR322-derived plasmids where head-on collision between the replication fork and the RNA polymerase transcribing the TetR gene was allowed or avoided. The results obtained indicate that this type of collision triggers knotting of the daughter duplexes behind the fork. We propose this deleterious topological consequence could explain the instability of pBR322 and could be also one of the reasons for nature to avoid head-on collision of transcription and replication.This work was partially supported by grants PM97-0138 and PGC PB98-048 from the Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT), 99/0850 from the Spanish Fondo de Investigación Sanitaria (FIS) and 08.6/0016/1997 from the Comunidad Autónoma de Madrid (CAM).Peer reviewe

Thymidine stress and nascent replicon maturation

1 p.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

The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks

14 p.-7 fig.In eukaryotes, ribosomal genes (rDNA) are organized in tandem repeats localized in one or a few clusters. Each repeat encompasses a transcription unit and a non-transcribed spacer. Replication forks moving in the direction opposite to transcription are blocked at specific sites called replication fork barriers (rRFBs) in the non-transcribed spacer close to the 3΄ end of the transcription unit. Here, we investigated and quantified the efficiency of rRFBs in Saccharomyces cerevisiae and to this end transfected budding yeast cells that express dissimilar quantities of Fob1 with circular minichromosomes containing different copies of the minimal 20-bp DNA segment that bind Fob1. To identify fork stalling we used high-resolution 2D agarose gel electrophoresis. The results obtained indicated that neighbor DNA sequences and the relative abundance of Fob1 modulate the efficiency of rRFBs to stall replication forks.Spanish Ministerio de Economía y Competitividad [BFU2014-56835 to J.B.S.]. Funding for open access charge: Spanish Ministerio de Economía y Competitividad [BFU2014-56835].Peer reviewe