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DNA replication dynamics in Trypanosoma cruzi: licensing and replication rate characterization

Grant number: 14/24170-5
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): June 01, 2015
Effective date (End): May 31, 2019
Field of knowledge:Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
Principal researcher:Maria Carolina Quartim Barbosa Elias Sabbaga
Grantee:Marcelo Santos da Silva
Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
Associated research grant:13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling, AP.CEPID
Associated scholarship(s):17/18719-2 - Structural analysis of the origin recognition complex (ORC) in Trypanosoma brucei using cryo-electron microscopy and single-particle analysis, BE.EP.PD

Abstract

Trypanosoma cruzi is a flagellated protozoa belonging to Trypanosomatidae family, and etiologic agent of Chagas's disease, a neglected disease that affects millions of people worldwide. To survive, these parasites require replicate their DNA adequately, which involves a coordinated action of many proteins. DNA replication starts with the licensing of a pre-replication machinery (pre-RC) in specific regions of DNA called origins of replication. The dynamics and the proteins involved in pre-RC assembly are not well established in trypanosomatids. Thus, we intend to identify and characterize potential TcOrc1/Cdc6 interactors, which would be responsible for the recognition and activation of replication origins in T. cruzi. To do this, we will perform capture assays (immunoprecipitation and co-immunoprecipitation), identify the molecules in SDS-PAGE and mass spectrometer, and confirm their role in replication by knockout. In parallel, we will perform pull down assays using TcOrc1/Cdc6 recombinant, marked with different tags (6xHis and MBP) to see if this protein oligomerizes. If so, this oligomerization will be confirmed by in vitro and in vivo assays. In addition, we also intend to evaluate how the DNA metabolic processes (transcription and repair) influence the replication rate in this parasite. In this direction, we will inhibit transcription (using ±-manitin), induce DNA damage (using UV radiation and H2O2) and check, through the SMARD (Single Molecule Analisys of Replicated DNA) technique, possible alterations in DNA replication rate. It is worth mentioning that this knowledge may assist future antiparasitary strategies, since DNA replication is related directly to chromosome maintenance and cell viability. (AU)

Scientific publications (21)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
COSTA-SILVA, HELLIDA MARINA; RESENDE, BRUNO CARVALHO; UMAKI, ADRIANA CASTILHOS SOUZA; PRADO, WILLIAN; DA SILVA, MARCELO SANTOS; VIRGILIO, STELA; MACEDO, ANDREA MARA; PENA, SERGIO DANILO JUNHO; TAHARA, ERICH BIRELLI; TOSI, LUIZ RICARDO ORSINI; ELIAS, MARIA CAROLINA; ANDRADE, LUCIANA OLIVEIRA; REIS-CUNHA, JOAO LUIS; FRANCO, GLORIA REGINA; FRAGOSO, STENIO PERDIGAO; MACHADO, CARLOS RENATO. DNA Topoisomerase 3 alpha Is Involved in Homologous Recombination Repair and Replication Stress Response in Trypanosoma cruzi. FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, v. 9, MAY 13 2021. Web of Science Citations: 0.
MARIN, PAULA ANDREA; OBONAGA, RICARDO; PAVANI, RAPHAEL SOUZA; DA SILVA, MARCELO SANTOS; DE ARAUJO, CHRISTIANE BEZERRA; LIMA, ANDRE ARRUDA; AVILA, CARLA CRISTI; CESTARI, IGOR; MACHADO, CARLOS RENATO; ELIAS, MARIA CAROLINA. ATR Kinase Is a Crucial Player Mediating the DNA Damage Response in Trypanosoma brucei. FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, v. 8, DEC 22 2020. Web of Science Citations: 0.
DA SILVA, MARCELO S. Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism. BIO-PROTOCOL, v. 10, n. 20 OCT 20 2020. Web of Science Citations: 0.
DE ARAUJO, CHRISTIANE BEZERRA; CHAGAS DA CUNHA, JULIA PINHEIRO; INADA, DAVI TOSHIO; DAMASCENO, JEZIEL; JERONIMO LIMA, ALEX RANIERI; HIRAIWA, PRISCILA; MARQUES, CATARINA; GONCALVES, EVONNILDO; NISHIYAMA-JUNIOR, MILTON YUTAKA; MCCULLOCH, RICHARD; ELIAS, MARIA CAROLINA. Replication origin location might contribute to genetic variability in Trypanosoma cruzi. BMC Genomics, v. 21, n. 1 JUN 22 2020. Web of Science Citations: 0.
DA SILVA, MARCELO SANTOS; LOPES, PRISCILA HESS; ELIAS, MARIA CAROLINA; TAMBOURGI, V, DENISE. Cytotoxic and genotoxic effects on human keratinocytes triggered by sphingomyelinase D from Loxosceles venom. ARCHIVES OF TOXICOLOGY, v. 94, n. 10 JUN 2020. Web of Science Citations: 1.
DA SILVA, MARCELO S.; VITARELLI, MARCELA O.; SOUZA, BRUNO F.; ELIAS, MARIA CAROLINA. Comparative Analysis of the Minimum Number of Replication Origins in Trypanosomatids and Yeasts. GENES, v. 11, n. 5 MAY 2020. Web of Science Citations: 0.
DA SILVA, MARCELO S.; CAYRES-SILVA, GUSTAVO R.; VITARELLI, MARCELA O.; MARIN, PAULA A.; HIRAIWA, PRISCILA M.; ARAUJO, CHRISTIANE B.; SCHOLL, BRUNO B.; AVILA, ANDREA R.; MCCULLOCH, RICHARD; REIS, MARCELO S.; ELIAS, MARIA CAROLINA. Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration. SCIENTIFIC REPORTS, v. 9, DEC 6 2019. Web of Science Citations: 0.
TEIXEIRA, SAMUEL COTA; LOPES, DAIANA SILVA; DA SILVA, MARCELO SANTOS; CORDERO DA LUZ, FELIPE ANDRES; CIRILO GIMENES, SARAH NATALIE; BORGES, BRUNA CRISTINA; DA SILVA, ALINE ALVES; MARTINS, FLAVIA ALVES; DOS SANTOS, MARIUS ALVES; TEIXEIRA, THAISE LARA; OLIVEIRA, RICARDO A.; RODRIGUES AVILA, VERIDIANA DE MELO; BARBOSA SILVA, MARCELO JOSE; ELIAS, MARIA CAROLINA; MARTIN, RENE; DA SILVA, CLAUDIO VIEIRA; KNOELKER, HANS-JOACHIM. Pentachloropseudilin Impairs Angiogenesis by Disrupting the Actin Cytoskeleton, Integrin Trafficking and the Cell Cycle. CHEMBIOCHEM, v. 20, n. 18 AUG 2019. Web of Science Citations: 0.
FERNANDES, CARLOS A. H.; PEREZ, ARINA M.; BARROS, ANDREA C.; DREYER, THIAGO R.; DA SILVA, MARCELO S.; MOREA, EDNA GICELA O.; FONTES, MARCOS R. M.; CANO, MARIA ISABEL N. Dual cellular localization of the Leishmania amazonensis Rbp38 (LaRbp38) explains its affinity for telomeric and mitochondrial DNA. Biochimie, v. 162, p. 15-25, JUL 2019. Web of Science Citations: 0.
COSTA, MONICA SOARES; GONCALVES, YASMIM GARCIA; TEIXEIRA, SAMUEL COTA; DE OLIVEIRA NUNES, DEBORA CRISTINA; LOPES, DAIANA SILVA; DA SILVA, CLAUDIO VIEIRA; DA SILVA, MARCELO SANTOS; BORGES, BRUNA CRISTINA; BARBOSA SILVA, MARCELO JOSE; RODRIGUES, RENATA SANTOS; RODRIGUES, VERIDIANA DE MELO; VON POELHSITZ, GUSTAVO; GERALDO YONEYAMA, KELLY APARECIDA. Increased ROS generation causes apoptosis-like death: Mechanistic insights into the anti-Leishmania activity of a potent ruthenium(II) complex. Journal of Inorganic Biochemistry, v. 195, p. 1-12, JUN 2019. Web of Science Citations: 0.
DE LIMA, LOYZE P.; CALDERANO, SIMONE G.; DA SILVA, MARCELO S.; DE ARAUJO, CHRISTIANE B.; VASCONCELOS, ELTON J. R.; IWAI, LEO K.; PEREIRA, CLAUDIO A.; FRAGOSO, STENIO P.; CAROLINA ELIAS, M. Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi. SCIENTIFIC REPORTS, v. 9, FEB 27 2019. Web of Science Citations: 1.
EMILIO TAVERNELLI, LUIS; MOTTA, MARIA CRISTINA M.; SILVA GONCALVES, CAMILA; DA SILVA, MARCELO SANTOS; ELIAS, MARIA CAROLINA; LUCIA ALONSO, VICTORIA; SERRA, ESTEBAN; CRIBB, PAMELA. Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity. SCIENTIFIC REPORTS, v. 9, JAN 17 2019. Web of Science Citations: 2.
DA SILVA, MARCELO S.; MARINI, PAULA A.; REPOLES, BRUNO M.; ELIAS, MARIA C.; MACHADO, CARLOS R. Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi. BIO-PROTOCOL, v. 8, n. 24 DEC 20 2018. Web of Science Citations: 0.
DA SILVA, MARCELO SANTOS; HOVEL-MINER, GALADRIEL A.; BRIGGS, EMMA M.; ELIAS, MARIA CAROLINA; MCCULLOCH, RICHARD. Evaluation of mechanisms that may generate DNA lesions triggering antigenic variation in African trypanosomes. PLOS PATHOGENS, v. 14, n. 11 NOV 2018. Web of Science Citations: 4.
SILVA, MAKSWELL ALMEIDA; LOPES, DAIANA SILVA; TEIXEIRA, SAMUEL COTA; CIRILO GIMENES, SARAH NATALIE; VASCONCELOS AZEVEDO, FERNANDA VAN PETTEN; POLLONI, LORENA; BORGES, BRUNA CRISTINA; DA SILVA, MARCELO SANTOS; BARBOSA, MARCELO JOSE; DE OLIVEIRA JUNIOR, ROBSON JOSE; ELIAS, MARIA CAROLINA; DA SILVA, CLAUDIO VIEIRA; GERALDO YONEYAMA, KELLY APARECIDA; RODRIGUES, VERIDIANA DE MELO; RODRIGUES, RENATA SANTOS. Genotoxic effects of BnSP-6, a Lys-49 phospholipase A(2) (PLA(2)) homologue from Bothrops pauloensis snake venom, on MDA-MB-231 breast cancer cells. International Journal of Biological Macromolecules, v. 118, n. A, p. 311-319, OCT 15 2018. Web of Science Citations: 3.
ALVES, CERES LUCIANA; REPOLES, BRUNO MARCAL; DA SILVA, MARCELO SANTOS; MENDES, ISABELA CECILIA; MARIN, PAULA ANDREA; NASCIMENTO AGUIAR, PEDRO HENRIQUE; SANTOS, SELMA DA SILVA; FRANCO, GLORIA REGINA; MACEDO, ANDREA MARA; JUNHO PENA, SERGIO DANILO; ANDRADE, LUCIANA DE OLIVEIRA; GUARNERI, ALESSANDRA APARECIDA; TAHARA, ERICH BIRELLI; ELIAS, MARIA CAROLINA; MACHADO, CARLOS RENATO. The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi. SCIENTIFIC REPORTS, v. 8, SEP 6 2018. Web of Science Citations: 2.
MARIN, PAULA ANDREA; DA SILVA, MARCELO SANTOS; PAVANI, RAPHAEL SOUZA; MACHADO, CARLOS RENATO; ELIAS, MARIA CAROLINA. Recruitment kinetics of the homologous recombination pathway in procyclic forms of Trypanosoma brucei after ionizing radiation treatment. SCIENTIFIC REPORTS, v. 8, MAR 29 2018. Web of Science Citations: 5.
DA SILVA, MARCELO S.; PAVANI, RAPHAEL S.; DAMASCENO, JEZIEL D.; MARQUES, CATARINA A.; MCCULLOCH, RICHARD; ORSINI TOSI, LUIZ RICARDO; ELIAS, MARIA CAROLINA. Nuclear DNA Replication in Trypanosomatids: There Are No Easy Methods for Solving Difficult Problems. Trends in Parasitology, v. 33, n. 11, p. 858-874, NOV 2017. Web of Science Citations: 9.
DA SILVA, MARCELO SANTOS; MARIN MUNOZ, PAULA ANDREA; ARMELIN, HUGO AGUIRRE; ELIAS, MARIA CAROLINA. Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids. Journal of Eukaryotic Microbiology, v. 64, n. 6, p. 756-770, NOV-DEC 2017. Web of Science Citations: 10.
DA SILVA, MARCELO SANTOS; SEGATTO, MARCELA; PAVANI, RAPHAEL SOUZA; GUTIERREZ-RODRIGUES, FERNANDA; BISPO, VANDERSON DA SILVA; GENNARI DE MEDEIROS, MARISA HELENA; CALADO, RODRIGO TOCANTINS; ELIAS, MARIA CAROLINA; NOGUEIRA CANO, MARIA ISABEL. Consequences of acute oxidative stress in Leishmania amazonensis: From telomere shortening to the selection of the fittest parasites. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, v. 1864, n. 1, p. 138-150, JAN 2017. Web of Science Citations: 9.
PAVANI, RAPHAEL SOUZA; DA SILVA, MARCELO SANTOS; HENRIQUE FERNANDES, CARLOS ALEXANDRE; MORINI, FLAVIA SOUZA; ARAUJO, CHRISTIANE BEZERRA; DE MATTOS FONTES, MARCOS ROBERTO; SANT'ANNA, OSVALDO AUGUSTO; MACHADO, CARLOS RENATO; CANO, MARIA ISABEL; FRAGOSO, STENIO PERDIGAO; ELIAS, MARIA CAROLINA. Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi. PLoS Neglected Tropical Diseases, v. 10, n. 12 DEC 2016. Web of Science Citations: 5.

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