Single molecule analysis of Trypanosoma brucei DNA replication dynamics
Nucleic Acids Research
Single molecule analysis of Trypanosoma brucei DNA replication dynamics
Simone Guedes Calderano 1 2
William C. Drosopoulos 0
Marina Moˆ naco Quaresma 1 2
Catarina A. Marques 3
Settapong Kosiyatrakul 0
Richard McCulloch 3
Carl L. Schildkraut 0
Maria Carolina Elias 1 2
0 Department of Cell Biology, Albert Einstein College of Medicine , Bronx, NY 10461 , USA
1 Center of Toxins, Immune Response and Cell Signaling - CeTICS, Instituto Butantan , S a ̃o Paulo, SP 05503-900 , Brasil
2 Laborat o ́rio Especial de Ciclo Celular, Instituto Butantan , S a ̃o Paulo, SP 05503-900 , Brasil
3 The Wellcome Trust Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Infection , Immunity and Inflammation , University of Glasgow , Glasgow, G128TA , UK
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 of multigenic 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.
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Chromosomal replication is initiated through the
assembly of the pre-replication complex (pre-RC) at DNA sites
called origins of replication (1). Unlike in most bacteria,
where the bi-directional DNA replication forks emerging
from a single replication origin duplicate the entire circular
genome, the genomes of eukaryotic cells are replicated from
many replication origins distributed along multiple
chromosomes. However, the features that define these origins
are not well understood in most eukaryotes. Well-defined,
conserved sequences that determine the sites of initiation
of DNA replication have been identified in Saccharomyces
cerevisiae (2), where origins are called autonomously
replicating sequences. In other eukaryotes consensus sequences
have not been described at mapped replication origins,
suggesting they can be defined by other parameters, such as
nucleosome positioning, histone modifications and 3-D
organization of the nucleus (3). The origins distributed
throughout eukaryotic genomes display variations in terms of
efficiency, including the frequency at which an origin fires
within a population of cells and activation time during
Sphase. Among this range of efficiency are dormant origins,
which are typically silent, but can be activated as backup
origins when passive replication from nearby origins is
impeded (4–6). Such origins may be crucial, because the
replication fork must overcome numerous natural obstacles,
including transcription complexes engaged along the same
DNA template (7–9).
Trypanosoma brucei is the etiological agent of
sleeping sickness, which primarily affects human populations in
some of the least developed countries of Central Africa.
During its life cycle, T. brucei alternates between insect and
mammalian hosts and the life cycle of this parasite includes
forms that are able to proliferate and forms that do not
replicate (10). Since infective forms are not able to
replicate their DNA, blockage of DNA replication may play a
role in T. brucei transmission. The 26-megabase genome of
T. brucei was sequenced in 2005 (11), in conjunction with
two other related kinetoplastid parasites, revealing a highly
unusual genomic organization, with each chromosome
containing directional gene clusters (DGCs) comprising an
average of 50 (but sometimes hundreds) of genes (12). Most
DGCs are transcribed (probably constitutively) by RNA
polymerase II, producing polycistronic transcripts that are
processed into single-gene mRNAs through trans-splicing
(13,14). Nuclear DNA replication in T. brucei and its
relatives has only begun to be characterized. Our group
identified a component of the pre-RC, the initiator machinery
present at replication origins, of T. brucei. By its homology
with the two pre-RC components Orc1 and Cdc6, we
designated it O (...truncated)