REPLICATION ORIGINS IN TRYPANOSOMES Ph1(1-2):Replication origins in trypanosomes and their position into nucleosomes during T.cruzi life cycle Chromosomal replication initiates with the assembly of the pre-replication complex(pre-RC) at DNA sites along the chromosomes that are called origins of replication1. The genomes of the majority of eukaryotic cells are replicated from many replication origins during the synthetic (S) phase of the cell cycle. This process requires that cells must ensure that sufficient numbers of origins are used in each S phase without the reuse of any origin in a single cell cycle. Unlike other eukaryotes, little is known about the DNA replication process in trypanosomes, protozoan parasites that appear early in the evolution. Using high throughput analysis of nascent DNA strand, this project intends to identify replication origins in T.cruzi and T.brucei, etiological agents of Chagas' and Sleeping Sickness diseases, respectively. Some obtained sequences will be checked as indeed replication origins using SMARD and ChIP assays. Ph2(3-4): Replication origins in trypanosomes and their position into nucleosomes during T.cruzi life cycle. The life cycle of T.cruzi alternates between replicative and non-replicative stages. The molecular bases that control the lack of DNA replication in non-replicative stages remain to be determined. It has already been showed that in eukaryotes replication origins are located in non-nucleosomal regions. Therefore, once identified replication origins in T.cruzi, we intend to investigate how these sequences are organized,concerning nucleosomal position, in different stages of T.cruzi life cycle. In this way, genomic DNAs will be digested with nuclease micrococcus, that digests all nucleosomal-free DNA, and nucleosomal protected DNA will be sequenced. Ph3(5): Characterization of trypanosomes DNA pre-replication machinery Chromosomal replication initiates with the assembly of the pre-replication complex (pre-RC) at DNA sites along the chromosomes that are called origins of replication1. In eukaryotes, the pre-RC is composed of an origin recognition complex (ORC) containing the Orc1-Orc6 molecules, two proteins named Cdc6 and Cdt1, and the mini-chromosome maintenance(MCM) complex, which is composed of Mcm2-Mcm7 molecules. As long as the pre-RC composed of ORC1-6, Cdc6, Cdt1, and MCM2-7 is organized on the chromatin, origins become licensed to replicate. In addition, other proteins must associate with the origin prior to the successful initiation of DNA synthesis. The binding of regulatory factors and components of the replication fork to DNA allows origin unwinding,the recruitment of replicative DNA polymerases, and finally the establishment of replication fork 2,3. Unlike other eukaryotes, little is known about the DNA replication process in trypanosomes, protozoan parasites that appear early in the evolution. As trypanosomatids have peculiar characteristics, with their genes transcribed in polycistronic units and processed by a trans-splicing reaction, and with gene expression controlled mainly at the post-transcriptional level4-6, we hypothesize that new strategies to deal with the regulation of replication can be found in these organisms. Genomic databases of trypanosomatids show that these organisms contain all MCM molecules, but they do not contain sequences in their genome that could code for the ORC subunits, Cdc6, or Cdt1. Trypanosomes have a gene for only 1 of the 6 subunits of the ORC, Orc1, which is also homologous to Cdc6. It is annotated as Orc17 and we named it Orc1/Cdc6. Previous results from our group showed that Orc1/Cdc6 is indeed a pre-replication machinery component that might be involved in the selection of replication origins in these organisms8. Using genetic reverse techniques (RNAi, expression of tag proteins), and conventional molecular and cellular assays, we intend to (i) search for molecules that could be components of the pre-replication machinery and/or (ii) understand how is the recruitment of MCM helicase complex directly by Orc1/Cdc6.
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