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. 2016 Dec 22;7:2041. doi: 10.3389/fmicb.2016.02041

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Copyright © 2016 Chiurillo, Moraes Barros, Souza, Marini, Antonio, Cortez, Curto, Lorenzi, Schijman, Ramirez and da Silveira.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Analysis of double-strand break repair events. (A) Schematic representation of PCR strategy used to amplify a 2.8-kb fragment of pTAC-D6C^∗I-SceI sequence spanning the I-SceI site in mutant T. cruzi epimastigotes. Fragments amplified with primers F1 and R1 were cloned into pGEM-T, and a nested PCR based on primers F2 and R2 was used to sequence the DSB region. (B) Summary table showing molecular constructs present in cloned cell lines of T. cruzi transfected-epimastigotes. (C) Nucleotide sequence alignment of the region adjacent to the DSB site in the wild-type T. cruzi telomeric sequence cloned into BAC-D6C with the D6C^∗I-SceI sequence including the I-SceI restriction site (in blue) (1); pTAC-D6C^∗I-SceI vector with pTAC-D6C^∗I-SceI mutant clone F5 (2); pTAC-D6C^∗ vector with pTREX-(SV40)I-SceI-GFP/pTAC-D6C^∗ mutant clone H9 (3); pTAC-D6C^∗I-SceI vector with pTREX-(SV40)I-SceI-GFP/pTAC-D6C^∗I-SceI mutant clones C12, D3, F10 and H7 (4); pTAC-D6C^∗I-SceI vector with pTREX-(SV40)I-SceI-GFP/pTAC-D6C^∗I-SceI mutant clone C8 (5). (D) Representation of DSB repair events by homologous recombination (HR). Recombination between regions flanking the I-SceI site in the pTAC-D6C^∗I-SceI molecule may have led to loss of the I-SceI site and replacement of this site with genomic sequences. Schemas (1,2) are representations of events shown in (B).