DNA replication in trypanosome: characterization of DNA replication forks and identification of DNA replication origins in Trypanosoma brucei

Abstract

DNA replication initiates with the assembly of the prereplication complex at DNA sites along chromosomes that are called origins of replication. In eukaryotes, these replication origins are well established only in S. Cerevisiae. Moreover, molecular bases that determine the timing of the firing of different replication origins along the S phase of the cell cycle are not determined. Therefore, the characterization of DNA replication in different eukaryote organisms is important for the knowing of the biology of this organism as well as of the biology of DNA replication. Here we intend to analyze single DNA molecules from Trypanosoma brucei in order to characterize: (i) the speedy and direction of replication forks, (ii) the localization and frequency of replication origins in specific DNA fragments. To get this goal we will use a methodology named SMARD (Single Molecule Analysis of Replicated DNA). In this methodology, two consecutives pulses of distinct thymidine analogous label DNA molecules. DNA is then extracted, digested with specific restriction enzymes and specific fragments are then isolated by pulse field gel electrophoresis (PFGE). Molecules are then stretched on slides and hybridized with specific probes in order to identify the molecules and the orientation of DNA fiber. This methodology allows the visualization of replication origins, replication termination and DNA replication forks. We intend to analyze three fragments of about 300 kb that together correspond to the 1Mb chromosome I from T. brucei. W e will analyze if the speed of DNA replication fork is constant along the entire chromosome and moreover, we intend to evaluate the number and localization of replication origins along the chromosome I. Finally, Dr. Richard McCulloch (University of Glasgow), identified sequences along T. brucei chromosome IV that interact with the T. brucei prereplication complex component Orc1/Cdc6. Therefore, this project intends also validate, using SMARD assay, which of these sequences are in fact replication origins. In conclusion, this project intends to investigate the molecular mechanisms of DNA replication in T. brucei, establishing the SMARD assay, a new methodology that is not being performed in Brazilian labs. (AU)