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H2020Individual fellowship2017–2019

RECREPEMLE · Recombination-derived replication in the eukaryotic microbe Leishmania: a genome-wide process?

Horizon 2020 — Marie Skłodowska-Curie Actions

Duration
2017-10-01 → 2019-09-30
EU contribution
€183,455
Participants
1
Scheme
MSCA-IF-EF-ST

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Results in brief

Recombination-derived replication in the eukaryotic microbe Leishmania: a genome-wide process?

Currently, more than 1 billion people are living in endemic areas at risk of infection with Leishmania, the agent of leishmaniases, with more than 20,000 deaths every year (https://www.who.int/leishmaniasis/en/). Effective treatment of the 300 thousand new leishmaniasis cases each year is challenging due to resistance to available drugs. Resistant isolates frequently present genomic alterations, including chromosome and gene amplification and/or loss (copy number variation; CNV), indicating that genome plasticity is a driver in drug resistance. Therefore, uncovering the mechanisms of genome maintenance and transmission in Leishmania, which remains poorly understood, is crucial to foster the development of new and more effective anti-leishmaniasis therapies. Whole genome replication initiation mapping using Marker Frequency Analysis coupled with deep sequencing (MFAseq) revealed that in Leishmania only a single high efficiency bi-directional origin is detected per chromosome in early S phase. Such origin singularity has not been seen in any other eukaryote and reveals a paradox: measurements of replication fork rate (~2.4 – 2.6 kb/min) and S phase duration (~3 hrs) in Leishmania suggest a single origin is insufficient for complete, timely genome replication. These suggest that unusual DNA replication mechanisms are explored by this parasite during genome transmission. The objective of this proposal was to test the innovative hypothesis that normal genome replication in Leishmania relies on, in addition to high efficiency locus-specific origins, stochastic origin-independent, homologous recombination (HR)-mediated initiation events. To test this, I have performed genome-wide analysis of replication dynamics and uncovered an unconventional replication initiation program, including DNA synthesis outside S phase. Also, I have evaluated the involvement of six different HR factors and RNAseH1, a R-loop resolving enzyme, with DNA replication and demonstrated that Rad51 and RNAseH1 are essential to the maintenance of the replication program in this parasite.

Data: CORDIS, © European Union

Project objective

In any organism, the need to reproduce is paramount, with genome replication being the earliest step in the process. The machinery and mechanisms of DNA replication are widely conserved, with initiation occurring at defined bi-directional DNA synthesis sites termed origins. Though origins may not be conserved DNA sequences, they are defined through binding conserved replication initiator factors. In some circumstances, such as after DNA damage or origin deletion, origin-independent replication can be observed in cellular organisms and can be driven by recombination. However, origin-independent replication is not normally considered to be a genome-wide, central reaction of genome replication. Very recently, genome-wide mapping of DNA replication initiation in two species of the kinetoplastid parasite Leishmania revealed only a single locus-specific origin per chromosome. Analyses of Leishmania S phase length and DNA synthesis speed suggest that a single origin-based strategy is insufficient for complete genome duplication, indicating replication in these eukaryotic microbes relies on origin-independent initiation to support origin-directed initiation. In this proposal, I will test the hypothesis that stochastic origin-independent DNA synthesis initiation events occur throughout the Leishmania genome due to homologous recombination acting on DNA lesions. If correct, this work will reveal the first example of a cellular organism that uses origin-independent replication as a core feature of genome duplication. The genome-wide use of recombination-driven replication would alter our view of DNA replication evolution and would explain the remarkable genome plasticity of Leishmania, with implications for how the parasite adapts genome structure and gene expression in the face of changing environments. Such adaptive change is seen during acquisition of resistance to anti-leishmanial drugs, and so this work will provide insight into the use and development of therapies.

Original text from CORDIS.

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Data: CORDIS, © European Union