Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi

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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

• Document type: Journal article
• Source: PLoS Neglected Tropical Diseases; v. 10, n. 12 DEC 2016.
• Web of Science Citations: 5

Abstract

Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi. (AU)

• FAPESP's process: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling
• Grantee: Hugo Aguirre Armelin
• Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 14/02978-0 - Functional analysis of RPA complex in Trypanosoma cruzi and its involvement with telomeric DNA
• Grantee: Raphael Souza Pavani
• Support type: Scholarships in Brazil - Doctorate
• FAPESP's process: 13/17864-8 - Structural Studies with Neurotoxic Phospholipases A2.
• Grantee: Carlos Alexandre Henrique Fernandes
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 14/13375-5 - Replication origins in trypanosomes
• Grantee: Christiane Bezerra de Araujo
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 15/10580-0 - Characterization of intra-S checkpoint in Trypanosoma cells
• Grantee: Maria Carolina Quartim Barbosa Elias Sabbaga
• Support type: Regular Research Grants
• FAPESP's process: 14/24170-5 - DNA replication dynamics in Trypanosoma cruzi: licensing and replication rate characterization
• Grantee: Marcelo Santos da Silva
• Support type: Scholarships in Brazil - Post-Doctorate