Dynamics of DNA replication in Trypanosoma cruzi: consequences of replicative stress

Abstract

Trypanosoma cruzi is the etiologic agent of Chagas disease, also known as American trypanosomiasis. This disease is transmitted mainly by vector disease and estimated that 6 to 7 million people worldwide, a majority in Latin America, are infected with the parasite. Despite all research efforts with the Chagas Disease and much knowledge acquired, there is still no vaccine or treatment that reduces the damage caused by T. cruzi in the human body. Thus, understanding the biology of the parasite opens doors to new therapies. In this context, our research group has been trying to understand DNA replication in trypanosomes, and the consequences of replicative stress. This project aims to investigate how T. cruzi responds to replicative stress to verify whether this response is related to the genetic plasticity presented by this parasite. More specifically, we will verify the presence of potential dormant origins, the ability of T. cruzi to activate dormant origins in the face of replicative stress, and the generation of mutations in situations of conflict between transcription-replication machinery, when these conflicts are co-directional or frontal. The identification of licensed origins in the genome will by the Chip-seq assay through the precipitation of the Orc1Cdc6 protein. Through mathematical modeling that considers the location of activated origins and the size of the chromosomes, we will infer regions of the chromosome with a greater probability of triggering dormant origins. Subsequently, we will experimentally validate in cell culture, induced to replicative stress with hydroxyurea, by RT-PCR these predictions of post-stress activated dormant origins. Furthermore, we will induce an origin of replication at a known point in the genome, seeking to verify the generation of mutations in the conflict between transcription-replication machinery. To do so, we will use the Orc1Cdc6 protein fused to the dead Cas9 protein, which recognizes a sequence in the genome but cannot cleave the DNA. Then, we will take the ORC complex to a specific point in the genome and verify, by nanosequencing of BrdU-tagged DNA, if this region has become an active origin. Subsequently, assembly of this system of known origin, will then be maintained for different times in the cell culture. Then, the polycistron containing genes that collided in a frontal (CF) and co-directional (CC) way will be sequenced to verify mutations.