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Alan D Grossman - One of the best experts on this subject based on the ideXlab platform.

  • identification of a single strand Origin of Replication in the integrative and conjugative element icebs1 of bacillus subtilis
    PLOS Genetics, 2015
    Co-Authors: Laurel D Wright, Christopher M Johnson, Alan D Grossman
    Abstract:

    We identified a functional single strand Origin of Replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal Replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle Replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle Replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso's in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand Origin of Replication, that these ICEs likely undergo autonomous Replication, and that Replication contributes to the stability and spread of these elements.

  • the sporulation protein sira inhibits the binding of dnaa to the Origin of Replication by contacting a patch of clustered amino acids
    Journal of Bacteriology, 2011
    Co-Authors: Lilah Rahnlee, Alan D Grossman, Houra Merrikh, Richard Losick
    Abstract:

    Bacteria regulate the frequency and timing of DNA Replication initiation by controlling the activity of the Replication initiator protein DnaA. SirA is a recently discovered regulator of DnaA in Bacillus subtilis whose synthesis is turned on at the start of sporulation. Here, we demonstrate that SirA contacts DnaA at a patch of 3 residues located on the surface of domain I of the Replication initiator protein, corresponding to the binding site used by two unrelated regulators of DnaA found in other bacteria. We show that the interaction of SirA with domain I inhibits the ability of DnaA to bind to the Origin of Replication. DnaA mutants containing amino acid substitutions of the 3 residues are functional in Replication initiation but are immune to inhibition by SirA.

  • Ordered association of helicase loader proteins with the Bacillus subtilis Origin of Replication in vivo
    Molecular Microbiology, 2010
    Co-Authors: Wiep Klaas Smits, Alexi I. Goranov, Alan D Grossman
    Abstract:

    The essential proteins DnaB, DnaD and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA Replication, and for Replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in Replication restart. During Replication initiation in vivo, it is not known if these proteins, and the Replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order. We used temperature-sensitive mutants or a conditional degradation system to inactivate each protein and test for association of the other proteins with oriC in vivo. We found that there was a clear order of stable association with oriC; DnaA, DnaD, DnaB, and finally DnaI-mediated loading of helicase. The loading of helicase via stable intermediates resembles that of eukaryotes and the established hierarchy provides several potential regulatory points. The general approach described here can be used to analyse assembly of other complexes.

Matthew K Waldor - One of the best experts on this subject based on the ideXlab platform.

  • molecular dissection of the essential features of the Origin of Replication of the second vibrio cholerae chromosome
    Mbio, 2015
    Co-Authors: Michael C Chao, Matthew A Gerding, Brigid M Davis, Matthew K Waldor
    Abstract:

    ABSTRACT Vibrionaceae family members are interesting models for studying DNA Replication initiation, as they contain two circular chromosomes. Chromosome II (chrII) Replication is governed by two evolutionarily unique yet highly conserved elements, the Origin DNA sequence oriCII and the initiator protein RctB. The minimum functional region of oriCII , oriCII-min , contains multiple elements that are bound by RctB in vitro , but little is known about the specific requirements for individual elements during oriCII initiation. We utilized undirected and site-specific mutagenesis to investigate the functionality of mutant forms of oriCII-min and assessed binding to various mutant forms by RctB. Our analyses showed that deletions, point mutations, and changes in RctB target site spacing or methylation all impaired oriCII-min -based Replication. RctB displayed a reduced affinity for most of the low-efficacy Origins tested, although its characteristic cooperative binding was generally maintained. Mutations that removed or altered the relative positions of Origin components other than RctB binding sites (e.g., AT-rich sequence, DnaA target site) also abolished replicative capacity. Comprehensive mutagenesis and deep-sequencing-based screening (OriSeq) allowed the identification of a previously uncharacterized methylated domain in oriCII that is required for Origin function. Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII . IMPORTANCE The genome of the enteric pathogen Vibrio cholerae consists of two chromosomes. While the chromosome I Replication Origin and its cognate Replication initiator protein resemble those of Escherichia coli, the factors responsible for chromosome II Replication initiation display no similarity to any other known initiation systems. Here, to enhance our understanding of how this DNA sequence, oriCII , and its initiator protein, RctB, function, we used both targeted mutagenesis and a new random-mutagenesis approach (OriSeq) to finely map the oriCII structural features and sequences required for RctB-mediated DNA Replication. Collectively, our findings reveal the extraordinary evolutionary honing of the architecture and motifs that constitute oriCII and reveal a new role for methylation in oriCII -based Replication. Finally, our findings suggest that the OriSeq approach is likely to be widely applicable for defining critical bases in cis -acting sequences.

  • independent control of Replication initiation of the two vibrio cholerae chromosomes by dnaa and rctb
    Journal of Bacteriology, 2006
    Co-Authors: Stephane Duigou, Kristine Groth Knudsen, Ole Skovgaard, Elisabeth S Egan, Anders Lobnerolesen, Matthew K Waldor
    Abstract:

    Although the two Vibrio cholerae chromosomes initiate Replication in a coordinated fashion, we show here that each chromosome appears to have a specific Replication initiator. DnaA overproduction promoted overinitiation of chromosome I and not chromosome II. In contrast, overproduction of RctB, a protein that binds to the Origin of Replication of chromosome II, promoted overinitiation of chromosome II and not chromosome I.

Yan Dong - One of the best experts on this subject based on the ideXlab platform.

  • new noncoding lytic transcripts derived from the epstein barr virus latency Origin of Replication orip are hyperedited bind the paraspeckle protein nono p54nrb and support viral lytic transcription
    Journal of Virology, 2015
    Co-Authors: Walter N Moss, Tina Ogrady, Monica Concha, Michael J Strong, Xia Wang, Yi Yu, Melody Baddoo, Kun Zhang, Claire Fewell, Yan Dong
    Abstract:

    ABSTRACT We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency Origin of Replication ( oriP ) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward (oriPtRs) transcripts are largely localized in the nucleus. While the oriPtLs are most likely noncoding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5′ untranslated regions may partially serve noncoding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics, and their expression is inhibited by phosphonoacetic acid. RNA sequencing (RNA-seq) analysis showed that oriPtLs and oriPtRs exhibited extensive “hyperediting” at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts, suggesting that oriPts interact with the paraspeckle-based innate antiviral immune pathway. Knockdown and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA Replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade. IMPORTANCE Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long noncoding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions, although these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency Origin of Replication. We show that they are hyperedited, that they interact with a relatively newly appreciated antiviral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic Replication.

Matthew J Mahon - One of the best experts on this subject based on the ideXlab platform.

  • vectors bicistronically linking a gene of interest to the sv40 large t antigen in combination with the sv40 Origin of Replication enhance transient protein expression and luciferase reporter activity
    BioTechniques, 2011
    Co-Authors: Matthew J Mahon
    Abstract:

    The simian virus 40 large T antigen (SVLT) induces Replication of plasmids bearing the SV40 Origin of Replication (SV40 ori) within mammalian cells. The internal ribosomal entry site (IRES) is an element that allows for the cotranslation of proteins from one polycistronic mRNA. Through the combination of these elements, IRES-dependent coexpression of a protein of interest and the SVLT, either constitutive or regulated, on plasmids bearing the SV40 ori generates a positive feedback loop, resulting in enhanced expression. A vector linking red fluorescent protein (RFP) to the IRES-SVLT element enhances fluorescence ∼10-fold over that demonstrated from a vector lacking this element. In transfection-resistant CV-1 cells, the RFP-IRES-SVLT vector substantially increases the number of cells expressing detectable levels of RFP. Furthermore, inclusion of the IRES-SVLT/SV40 ori elements in standard luciferase-based reporter gene constructs and associated effectors results in marked increases in luminescent output a...

  • vectors bicistronically linking a gene of interest to the sv40 large t antigen in combination with the sv40 Origin of Replication enhance transient protein expression and luciferase reporter activity
    BioTechniques, 2011
    Co-Authors: Matthew J Mahon
    Abstract:

    The simian virus 40 large T antigen (SVLT) induces Replication of plasmids bearing the SV40 Origin of Replication (SV40 ori) within mammalian cells. The internal ribosomal entry site (IRES) is an element that allows for the cotranslation of proteins from one polycistronic mRNA. Through the combination of these elements, IRES-dependent coexpression of a protein of interest and the SVLT, either constitutive or regulated, on plasmids bearing the SV40 ori generates a positive feedback loop, resulting in enhanced expression. A vector linking red fluorescent protein (RFP) to the IRES-SVLT element enhances fluorescence ~10-fold over that demonstrated from a vector lacking this element. In transfection-resistant CV-1 cells, the RFP-IRES-SVLT vector substantially increases the number of cells expressing detectable levels of RFP. Furthermore, inclusion of the IRES-SVLT/SV40 ori elements in standard luciferase-based reporter gene constructs and associated effectors results in marked increases in luminescent output and sensitivity, using the β-catenin/TCF pathway and the mammalian two-hybrid assay as models. Ultimately, vector systems combining these well-established elements (IRES-SVLT/SV40 ori) will increase the utility of transient transfection for the production of recombinant proteins, the use of transfection-resistant cell lines, and the effectiveness of luciferase-based high-throughput screening assays.

Jose Maria Carazo - One of the best experts on this subject based on the ideXlab platform.

  • large t antigen on the simian virus 40 Origin of Replication a 3d snapshot prior to dna Replication
    The EMBO Journal, 2003
    Co-Authors: Maria G Gomezlorenzo, Mikel Valle, Joachim Frank, Claudia Gruss, Carlos Oscar S. Sorzano, Xiaojiang S. Chen, Luis Enrique Donate, Jose Maria Carazo
    Abstract:

    Large T antigen is the replicative helicase of simian virus 40. Its specific binding to the Origin of Replication and oligomerization into a double hexamer distorts and unwinds dsDNA. In viral Replication, T antigen acts as a functional homolog of the eukaryotic minichromosome maintenance factor MCM. T antigen is also an oncoprotein involved in transformation through interaction with p53 and pRb. We obtained the three‐dimensional structure of the full‐length T antigen double hexamer assembled at its Origin of Replication by cryoelectron microscopy and single‐particle reconstruction techniques. The double hexamer shows different degrees of bending along the DNA axis. The two hexamers are differentiated entities rotated relative to each other. Isolated strands of density, putatively assigned to ssDNA, protrude from the hexamer–hexamer junction mainly at two opposite sites. The structure of the T antigen at the Origin of Replication can be understood as a snapshot of the dynamic events leading to DNA unwinding. Based on these results a model for the initiation of simian virus 40 DNA Replication is proposed.