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

Grant number: 14/24170-5
Support Opportunities: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
Acordo de Cooperação: Coordination of Improvement of Higher Education Personnel (CAPES)
Principal Investigator:Maria Carolina Quartim Barbosa Elias Sabbaga
Grantee:Marcelo Santos da Silva
Host 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)

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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)
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.; et al. 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, . (15/10580-0, 17/07693-2, 17/18719-2, 16/17775-3, 14/24170-5, 16/50050-2, 13/07467-1, 14/13375-5)
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, . (13/07467-1, 14/24170-5, 17/18719-2)
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; et al. 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, . (14/24170-5)
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, . (14/24170-5, 15/18641-8)
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; et al. Replication origin location might contribute to genetic variability in Trypanosoma cruzi. BMC Genomics, v. 21, n. 1, . (13/07467-1, 14/24170-5, 16/50050-2, 14/13375-5)
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, . (13/07467-1, 14/24170-5, 16/50050-2, 17/18719-2, 17/07693-2)
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, . (17/18719-2, 18/12364-0, 15/10580-0, 14/02978-0, 14/24170-5, 19/01895-8, 16/50050-2, 14/13375-5)
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; et al. 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, . (14/24170-5, 18/14398-0, 13/07467-1, 16/50050-2)
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; et al. 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, . (14/13375-5, 14/24170-5, 13/17864-8, 14/02978-0, 13/07467-1, 15/10580-0)
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, . (16/50050-2, 14/24170-5, 15/10580-0, 13/07467-1)
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, . (16/50050-2, 14/24170-5, 15/10580-0, 13/07467-1)
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, . (14/24170-5, 15/10580-0, 13/07467-1)
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, . (13/07467-1, 14/24170-5)
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, . (14/02978-0, 15/10580-0, 14/24170-5, 13/07467-1, 13/07937-8)
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; et al. The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi. SCIENTIFIC REPORTS, v. 8, . (16/50050-2, 13/07467-1, 14/24170-5)
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, . (16/50050-2, 17/18719-2, 13/07467-1, 14/24170-5)
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, . (15/17053-5, 13/07467-1, 14/24170-5)
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, . (16/50050-2, 14/13375-5, 13/07467-1, 14/24170-5)
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; et al. 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, . (13/07467-1, 14/24170-5)
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; et al. Pentachloropseudilin Impairs Angiogenesis by Disrupting the Actin Cytoskeleton, Integrin Trafficking and the Cell Cycle. CHEMBIOCHEM, v. 20, n. 18, . (13/07467-1, 14/24170-5)

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