Single molecule analysis of Trypanosoma brucei DNA replication dynamics

Simone Guedes Calderano, William C. Drosopoulos, Marina Mônaco Quaresma, Catarina A. Marques, Settapong Kosiyatrakul, Richard McCulloch, Carl L. Schildkraut, Maria Carolina Elias

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Abstract

Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genomewide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries ofmultigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated.

Original languageEnglish (US)
Pages (from-to)2655-2665
Number of pages11
JournalNucleic acids research
Volume43
Issue number5
DOIs
StatePublished - Mar 11 2015

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ASJC Scopus subject areas

  • Genetics

Cite this

Calderano, S. G., Drosopoulos, W. C., Quaresma, M. M., Marques, C. A., Kosiyatrakul, S., McCulloch, R., Schildkraut, C. L., & Elias, M. C. (2015). Single molecule analysis of Trypanosoma brucei DNA replication dynamics. Nucleic acids research, 43(5), 2655-2665. https://doi.org/10.1093/nar/gku1389