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

Periodic Reporting for period 1 - RECREPEMLE (Recombination-derived replication in the eukaryotic microbe Leishmania: a genome-wide process?)

Reporting period: 2017-10-01 to 2019-09-30

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.
In order to understand how DNA replication is coordinated in space and time during the cell cycle in Leishmania, I performed MFAseq analysis of cells during G1, early S, late S and G2/M. This analysis showed that the paucity of origins activated in early S phase is counterbalanced by DNA synthesis activity outside S phase. The core of the chromosomes is mainly replicated within S phase and are associated with simultaneous recruitment of acetylated histone H3 (AcH3), modified base J and the kinetochore factor KKT1. On the other hand, replication of the sub-telomeres occurs throughout the cell cycle including during G2/M and G1 and is associated with the recruitment of AcH3. We also show that subtelomeric DNA replication, unlike replication at the core, is highly sensitive to hydroxyurea and dependent on subunits of the 9-1-1 complex. Altogether, these analyses revealed the spatial, temporal and functional compartments of the replication program in this parasite.

To examine the contribution of HR factors to replication, I have generated 6 cell lines to perform DiCre-mediated conditional KO of 6 distinct HR-related factors. These includes RAD51 and its paralogues (Rad51_3, Rad51_4 and Rad51_6), which are predicted to catalyse strand invasion and exchange during HR. With this, I was able to demonstrate that these 4 factors are essential for long term survival of this parasite and that Rad51 and Rad51_3 are required for proper DNA synthesis. Moreover, genome wide mapping of replication initiation with MFAseq showed that absence of Rad51 leads to significant changes in the replication initiation program, revealing a central role of HR for DNA replication in this parasite.

I also generated cell lines for conditional KO of PIF6 and RNAseH1 to explore their roles in the replication program in Leishmania. PIF6 is a homologue of the PIF1 helicase, involved in a specialized type of HR called break induced replication (BIR). Absence of PIF did not result in any pronounced defect neither in growth or in replication. On the other hand, absence of RNAseH1, which is involved in the resolution of DNA: RNA hybrids (R-loops), resulted in substantial reconfiguration of the replication initiation program.

The results of the MSCA were disseminated through presentations at the following events:

- EMBL Symposia: DNA Replication: From Basic Biology to Disease on 07 - 10 May 2018. Heidelberg, Germany.
- International Scientific Advisory Board & WCMP Retreat on 29 - 31 May 2018. Glasgow, Scotland.
- XXXIV Annual Meeting of the Brazilian Society of Protozoology on 05 – 07 November 2018. Caxambu, Brazil.
- Research Update Meeting/Seminar Roster on 29 March 2019. Glasgow, Scotland.
- Scottish DNA Replication Network Meeting on 08 May 2019. Saint Andrews, Scotland.
- International Scientific Advisory Board & WCMP Retreat 28 - 30 May 2019. Glasgow, Scotland.
- Kinetoplastid Molecular Cell Biology Meeting on 27 April – 01 May 2019. Woods Hole, USA.
- Research Update Meeting/Seminar Roster on 18 October 2019. Glasgow, Scotland.
In any organism, the need to reproduce is paramount, with genome replication being the earliest step in the process. Analyses of Leishmania S phase length and DNA synthesis speed suggest that a single origin-based strategy, as previously suggested, is insufficient for complete genome duplication, indicating replication in these eukaryotic microbes relies on unusual DNA replication strategies.

The combined usage of CRSPR/Cas9 and DiCre allowed us not only to rapidly generate the desired cell lines, but also investigate the role of HR factors previously thought to be essential. This sets the framework for more efficient and precise conditional genome engineering in Leishmania, useful not only to examine genome maintenance-related factors but any other gene, including essential ones.

To our knowledge, this project is the first in depth dissection of the genome duplication program and the role HR factors in DNA replication in Leishmania parasites.

As we showed with this study, the genome-wide use of HR-driven replication further illustrate that DNA replication in Leishmania has evolved by exploring unconventional strategies.
Leishmaniasis can have drastic effects on human health, being a major burden in developing and tropical countries. Tackling leishmaniasis is therefore of paramount importance to foster the wellbeing and prosperity in these regions.

Ultimately, the results obtained during this MSCA 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 anti-leishmaniasis therapies.
How Leishmania Replicate Its DNA ?