Genome duplication in Leishmania major relies on persistent subtelomeric DNA replication

Abstract

DNA replication is needed to duplicate a cell's genome in S-phase and segregate it during cell division. Previous work in Leishmania detected DNA replication initiation at just a single region in each chromosome, an organisation predicted to be insufficient for complete genome duplication within S-phase. Here, we show that acetylated histone H3 (AcH3), base J and a kinetochore factor colocalise in each chromosome at only a single locus, which corresponds with previously mapped DNA replication initiation regions and is demarcated by localised G/T skew and G4 patterns. In addition, we describe previously undetected subtelomeric DNA replication in G2/M and G1 phase-enriched cells. Finally, we show that subtelomeric DNA replication, unlike chromosome-internal DNA replication, is sensitive to hydroxyurea and dependent on 9-1-1 activity. These findings indicate that Leishmania's genome duplication programme employs subtelomeric DNA replication initiation, possibly extending beyond S-phase, to support predominantly chromosome-internal DNA replication initiation within S-phase.

High genome plasticity and frequent genetic exchange in Leishmania tropica isolates from Afghanistan, Iran and Syria

“…In Leishmania, the maintenance of telomeres occurs through rolling circle replication via extrachromosomal amplification, as in Kluyveromyces lactis (84,85) rather than telomeric loop formation (77). And outside of the S cell cycle phase, DNA replication has been detected proximal to the chromosome telomeres, resembling a subtelomeric DNA synthesis activity (56). Our isolates had extensive aneuploidy and higher heterozygosity than previously reported (46).…”

Diverse telomeres in trypanosomatids

“…Variable telomere length in Leishmania spp. (and possibly other Leishmaniinae) may be explained by the presence of a stress-sensitive telomere-proximal replication activity outside S phase of the cell cycle in these species (Damasceno et al ., 2020, 2021).…”

Protein kinase signalling at the Leishmania kinetochore captured by XL-BioID

“…JBP2 and JBP3 are involved in synthesis and binding of base-J respectively, a glycosylated thymidine DNA base found only in kinetoplastids 20 . Base-J has been shown to be enriched at centromeres 21 , the finding that JBP2 and JBP3 are close to the kinetochore raise the possibility of base-J having a role in specifying the kinetochore binding site in Leishmania. The centromere in Leishmania is also the major origin of replication for chromosomes, explaining the enrichment of the origin of replication proteins Orc1, Orc4 and Orc5 22 .…”

“…We were able to endogenously tag both alleles of KKT3 with miniTurboID, since KKT3 is an essential protein kinase in Leishmania this indicated that the tag did not block protein function 27 . Parasites were synchronised with hydroxyurea at early S phase, then released to resume the cell cycle 21 . A 30 min biotinylation was performed at 0, 4 and 8 hrs following hydroxyurea release, which samples KKT3 proximal proteins and phosphosites at G1/S, S and G2/M stages respectively.…”

“…XL-BioID BirA*::BDF5, KKT2::BirA*, BirA*::KKT3, KKT19::BirA* parasites were grown to 4 x 10 6 ml -1 at which point biotinylation was initiated in 3 replicates of each line, by adding biotin to 150 μM for 18hrs. BDF5::mT and KKT3::mT parasites were grown to 2 x 10 6 ml -1 then synchronised by adding hydroxyurea to 0.4 mg ml -1 for 18hrs 21 . Synchronised parasites were washed twice in culture medium and resuspended at 4 x 10 6 ml -1 to proceed with the cell cycle, 5 replicates of each line and condition were used.…”