Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi.

Miguel A Chiurillo,Roberto R Moraes Barros,Renata T Souza,Hernán A Lorenzi,José F Da Silveira,Danielle R Cortez,María Á Curto,José L Ramirez,Cristiane R Antonio,Marjorie M Marini,Alejandro G Schijman

doi:10.3389/fmicb.2016.02041

Abstract

Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.

Highlights

Trypanosoma cruzi exhibits a vast repertoire of surface antigens encoded by ∼ 18% of all proteincoding genes, which are directly implicated in the parasite’s interaction with insect vectors and vertebrate hosts (El-Sayed et al, 2005; De Pablos and Osuna, 2012)
Despite the high level of polymorphism in T. cruzi subtelomeric regions, an organizational pattern frequently observed in T. cruzi chromosome ends is the presence of TS genes and pseudogenes flanked by retrotransposon hot spot (RHS) sequences (Kim et al, 2005; Moraes Barros et al, 2012), resembling the organization of the repetitive regions adjacent to variant surface glycoprotein (VSG) genes in T. brucei telomeres (Hertz-Fowler et al, 2008)
To import the I-SceI protein into the parasite nucleus, the nuclear localization signal (NLS) from SV40 large T antigen or T. cruzi histone H2B (TcH2B; Marchetti et al, 2000) was fused to the N-terminus of the I-SceI protein. (SV40)I-SceI open reading frame (ORF) was PCR amplified from the pLew100-NLSISceI-HA vector while the (TcH2B)I-SceI fusion gene was synthesized by GenScript (USA)

Summary

Introduction

Trypanosoma cruzi exhibits a vast repertoire of surface antigens encoded by ∼ 18% of all proteincoding genes, which are directly implicated in the parasite’s interaction with insect vectors and vertebrate hosts (El-Sayed et al, 2005; De Pablos and Osuna, 2012). Despite the high level of polymorphism in T. cruzi subtelomeric regions, an organizational pattern frequently observed in T. cruzi chromosome ends is the presence of TS genes and pseudogenes flanked by RHS sequences (Kim et al, 2005; Moraes Barros et al, 2012), resembling the organization of the repetitive regions adjacent to variant surface glycoprotein (VSG) genes in T. brucei telomeres (Hertz-Fowler et al, 2008) These (70-bp) repetitions are involved in recombination mechanisms responsible for antigenic variation in the African trypanosome (Boothroyd et al, 2009; Glover et al, 2013; Li, 2015)

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