title: Single molecule analysis of Trypanosoma brucei DNA replication dynamics.
abstract: Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated.
authors.affiliation: Laboratório Especial de Ciclo Celular, Instituto Butantan, São Paulo, SP 05503-900, Brasil Center of Toxins, Immune Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP 05503-900, Brasil.
authors.forename: Simone Guedes
authors.initials: SG
authors.lastname: Calderano
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Drosopoulos
authors.affiliation: Laboratório Especial de Ciclo Celular, Instituto Butantan, São Paulo, SP 05503-900, Brasil Center of Toxins, Immune Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP 05503-900, Brasil.
authors.forename: Marina Mônaco
authors.initials: MM
authors.lastname: Quaresma
authors.affiliation: The Wellcome Trust Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G128TA, UK.
authors.forename: Catarina A
authors.initials: CA
authors.lastname: Marques
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Settapong
authors.initials: S
authors.lastname: Kosiyatrakul
authors.affiliation: The Wellcome Trust Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G128TA, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: McCulloch
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA carol@butantan.gov.br.
authors.forename: Carl L
authors.initials: CL
authors.lastname: Schildkraut
authors.affiliation: Laboratório Especial de Ciclo Celular, Instituto Butantan, São Paulo, SP 05503-900, Brasil Center of Toxins, Immune Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, SP 05503-900, Brasil carol@butantan.gov.br.
authors.forename: Maria Carolina
authors.initials: MC
authors.lastname: Elias
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002875:Chromosomes / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics*; D005434:Flow Cytometry; D018503:Genome, Protozoan / Q000235:genetics; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D008018:Life Cycle Stages / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics*; D013997:Time Factors; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development
chemical_list: D018797:Cell Cycle Proteins; D016054:DNA, Protozoan; D019384:Nucleic Acid Synthesis Inhibitors; D051738:Origin Recognition Complex; D015800:Protozoan Proteins; D006918:Hydroxyurea