Single molecule analysis of Trypanosoma brucei DNA replication dynamics

Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromo-somes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiologi-cal agent of sleeping sickness, localized its replica-tion origins to the boundaries of multigenic transcrip-tion 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. bru-cei 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 pro-cyclic forms. We present evidence for replication ter-minating 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. Im-portantly, 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 find-ings support the existence of more replication ori-gins in T. brucei than previously appreciated