Conflict between replication and transcription: analysis of the replication fork in Trypanosoma brucei cells that present the transcription machinery stagnant in the genome

Abstract

Our lab studies DNA replication in trypanosomes, the etiological agents of Chagas disease (Trypanosoma cruzi) and sleeping sickness (Trypanosoma brucei). In this sense, we intend, first, to add information to the biology of these organisms rather peculiar, that diverged early in the eukaryotic lineage. Moreover, generate data that may be used in the prophylaxis and therapy of these diseases. Several lines of evidence suggest that there are conflicts between the complexes that replicate DNA and those that transcribe the DNA template. These conflicts can lead to blockage of replication and genomic instability. To circumvent these conflicts, cells protect themselves in two distinct ways. The first is preventing the replication and transcription machinery of be in the same DNA template. The second way is by counting on proteins capable of removing from the genome the stagnant transcription machinery in order to do not compromise the duplication of DNA and consequently the stability of the genetic material. This project aims to check if T. brucei replisome activity is compromised when the RNA polymerase is stalled in the genome. Thus, will be used the SMARD technique (Single-Molecule Analysis of Replicated DNA), already standardized in our laboratory. In this technique, the DNA molecules that are being replicated are marked by two successive pulses with different analogs of thymidine, allowing visualization of replication origins. More than that, it is possible to verify the extent of sister forks. When DNA is equally replicated in both directions from the origin of replication those forks are symmetrical molecules. It is also possible to infer the speed of the replication fork in the analyzed cells. Thus, the bloodstream form of T. brucei, which presents the CSB gene silenced by RNAi and therefore is not capable of removing the stagnant transcription machinery from the genome, will be compared with the control cell (normal expression of the gene CSB) for (I) average speed of the forks replication, and (II) the number of symmetrical replication forks to the asymmetrical replication forks (corresponding to compromised replissomes).(AU)