Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism

Laffitte, Marie-Claude N.; Leprohon, Philippe; Maripier, Hainse; Légaré, Danielle; Masson, Jean‐Yves; Ouellette, Marc

doi:10.1371/journal.pgen.1006117

Cited by 32 publications

(44 citation statements)

References 98 publications

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“…In contrast, the subtelomeric regions of intra and post S phase DNA synthesis can only be correlated with AcH3, and we cannot be sure that this form of chromatin is actually a determinant of replication initiation. One possibility is that subtelomeric DNA replication is not ORC-derived, but instead derives from DNA repair that is are required for the stability of sub telomeric regions, by either controlling copy number variation in these regions (76) or by directly driving replication initiation. Such an activity could be related to MiDAS, but may also differ since it appears to be a constitutive component of the Leishmania genome replication programme and looks like it could be related to what has been proposed as a strategy for telomere maintenance (37).…”

Section: Discussionmentioning

confidence: 99%

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

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Once every cell cycle, DNA replication takes place to allow cells to duplicate their genome and segregate the two resulting copies into offspring cells. In eukaryotes, the number of DNA replication initiation loci, termed origins, is proportional to chromosome size. However, previous studies have suggested that in Leishmania, a group of single-celled eukaryotic parasites, DNA replication starts from just a single origin per chromosome, which is predicted to be insufficient to secure complete genome duplication within S phase.Here, we show that the paucity of origins activated in early S phase is balanced by DNA synthesis activity outside S phase. Simultaneous recruitment of acetylated histone H3 (AcH3), modified base J and the kinetochore factor KKT1 is exclusively found at the origins used in early S phase, while subtelomeric DNA replication can only be linked to AcH3 and displays persistent activity through the cell cycle, including in G2/M and G1 phases. We also show that subtelomeric DNA replication, unlike replication from the previously mapped origins, is sensitive to hydroxyurea and dependent on subunits of the 9-1-1 complex.Our work indicates that Leishmania genome transmission relies on an unconventional DNA replication programme, which may have implications for genome stability in this important parasite. sequence read depth in the former relative to the latter. As result, DNA synthesis very late in S-phase, or that occurs outside of S-phase, would escape detection. To test this, we modified the MFA-seq approach in order that DNA content enrichment could be calculated in replicating cells relative to naturally occurring non-replicative cells. Mapping origins of replication by calculating DNA enrichment in exponentially growing cells versus cells in a stationary state has been used successfully in bacteria (42) and yeast (43). Thus, we reasoned that L. major in stationary phase could also serve as a non-replicative control for normalization in MFA-seq analysis compared with cells that are growing exponentially. To test this, we used flow cytometry to compare DNA content of exponentially growing and stationary phase cells and, in addition, compared the capacity of the two cell populations to incorporate the thymidine analogue 5-ethynyl-2'deoxyuridine (EdU; Fig. 1A,B). Flow cytometry showed that the proportion of cells in S phase (with DNA content between 1C and 2C) was substantially lower in a population of stationary cells compared with exponentially growing cells (Fig. 1A). Concomitantly, stationary phase cells, unlike exponentially growing cells, failed to incorporate EdU, even upon relatively long periods of incubation ( Fig. 1B). Thus, L. major cells in stationary phase do not perform any detectable DNA synthesis and were therefore deemed suitable to be used as the non-replicative sample in MFA-seq analysis.Next, we compared MFA-seq profiles using read depth ratios from FACS-sorted early S (ES) and G2 cells, and from exponentially growing cells (EXP) and stationary (STA) cells (Fig. 1C). As reported ...

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Section: Discussionmentioning

confidence: 99%

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

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“…RAD50, the largest component of the MRN complex, belongs to the structural maintenance of chromosomes (SMC) family of proteins 48 and has not been examined in T. brucei , though the gene has been reported to be essential in Leishmania infantum 49 . The domain architecture of RAD50 is approximately palindromic (Figure 1A) and characterized by the presence of ATP-binding cassette (ABC)-ATPase domains at the N- and C-termini, each followed by an MRE11 binding site (MBS), and then by anti-parallel coiled-coil regions, which form linker structures that enable the MRN complex to act as a tethering scaffold to hold broken chromosomes together for repair 50 .…”

Section: Resultsmentioning

confidence: 99%

“…The data shown here indicate that the role of T. brucei RAD50 in recognizing a DSB and initiating the DDR appears consistent with its role in other eukaryotes 64,65 . Previous characterization of MRE11 has revealed a role in HR and genomic stability in both T. brucei 46,47 and Leishmania 49,66 , suggesting RAD50 and MRE11 act together. Here we show that that MRE11 is essential for the DDR and HR and, in its absence, repair is predominantly achieved by MMEJ.…”

Section: Discussionmentioning

confidence: 97%

RAD50 promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

Glover

Mehnert

Hutchinson

et al. 2020

Preprint

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147 words 23 24 ABSTRACT 25 Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is 26 especially important for recombination of the subtelomeric variant surface glycoprotein during 27 antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to 28 homologous recombination through facilitating resection and governing the DNA damage response. 29The function of RAD50 in trypanosomes is untested. Here we report that RAD50 is required for 30 RAD51-dependent homologous recombination, phosphorylation of histone H2A and controlled 31 resection following a DNA double strand break (DSB). Perhaps surprisingly, DSB resection in the 32 rad50 nulls was not impaired and appeared to peak earlier than in the parental strains. Finally, we 33 show that RAD50 suppresses DNA repair using donors with short stretches of homology at a 34 subtelomeric locus, with null strains producing a greater diversity of expressed VSG variants following 35 DSB repair. We conclude that RAD50 promotes stringent homologous recombination at subtelomeric 36 loci and restrains antigenic variation. 37 38 42 vector, the tsetse fly, and mammalian hosts, where they colonise the blood, fat 1 and skin 2 and 43 eventually cross the blood brain barrier in late stage infection. If left untreated, trypanosomiasis is 44 normally fatal 3 . In the mammalian host, each trypanosome cell is covered in a dense layer of a single 45 species of variant surface glycoprotein (VSG). The highly immunogenic VSG layer 4,5 acts as an 46 barrier, concealing other surface components from the host immune response 6 . Trypanosomes 47 maintain a persistent infection by continuously escaping the host's immune response though antigenic 48 variation 7 . Central to this survival strategy is monoallelic expression of the VSG from a subtelomeric 49 locus, known as an expression site (VSG-ES), and stochastic VSG switching. The ~ 15 VSG-ESs in 50 the trypanosome genome share a high degree of sequence and structure conservation 8 , each being 51 an RNA polymerase-I (RNA Pol-I) polycistronic transcription unit with a single VSG gene found 52 adjacent to the telomere, up to 60 kb downstream of the promoter 8 . The VSG gene is flanked by two 53 sets of repetitive sequence: downstream is the telomere, and upstream is a block of repetitive 54 sequence, known as the 70-bp repeats, which modulates VSG switching 8,9 . Characteristic of a 55 trypanosome infection are recrudescent waves of parasitemia, each of which is composed of a diverse 56 VSG expressing population, with between 7 -79 VSGs detected in each peak of parasitemia 10-12 . 57 VSG diversity arises through altering the single VSG-ES that is transcribed or, more commonly, by 58 recombination of silent VSGs into the active VSG-ES. The seemingly unrestricted use of VSG genes 59 might be expected to result in a rapid exhaustion of the VSG gene repertoire. However, the parasite's 60 ability to sustain an infection appears to lie in an enormous repertoire of >2000 VSG genes ...

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“…Though we did not test for similar effects after RAD51-6 KO, similar outcomes might be expected. In addition, we did not attempt to test for larger genome changes, but translocations have been described in MRE11 (27), which can guide RAD51 function.…”

Section: Discussionmentioning

confidence: 99%

“…In T. cruzi, HR has been suggested to be a driver of variability in multigene families (24,25) and in cell hybridisation (26). Finally, in Leishmania, HR related factors have been implicated in mediating the formation or maintenance of episomes, which appear to form stochastically, arise genome-wide and have been implicated in acquisition of drug resistance (27)(28)(29)(30)(31)(32).…”

Section: Introductionmentioning

confidence: 99%

Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication

Damasceno

Reis-Cunha

Crouch

et al. 2019

Preprint

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Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genomewide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal, but in each case growth slows with time and leads to DNA damage, accumulation of cells with aberrant DNA content, and genome-wide mutation. Despite these similarities, we show that only loss of RAD51 and RAD51-3 impairs DNA synthesis, and that the factors act in distinct ways. Finally, we reveal that loss of RAD51 has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

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Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism

Cited by 32 publications

References 98 publications

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

RAD50 promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication

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