Ku80 is involved in telomere maintenance but dispensable for genomic stability in Leishmania mexicana

Abstract

Background Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. Methodology/Principal findings We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. Conclusion/Significance Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.

Reconstitution of Mycobacterium marinum Nonhomologous DNA End Joining Pathway in Leishmania

“…As shown in Fig. 4A , unlike what were reported in T. brucei and L. mexicana ( 43 – 45 ), no progressive telomere elongation or shortening was observed in WT or Ku70-deficient L. donovani cells. All the analyzed L. donovani cells retained similar lengths of telomeres over the 9-week culture period.…”

“…In addition, Ku70/80 proteins are required for the normal healthy cell proliferation. However, unlike in other organisms ( 43 – 45 ), the normal lengths of the telomeres are maintained in Ku-deficient L. donovani cells. We also introduced Mycobacterium marinum two components (Ku and Ligase D) NHEJ pathway into Leishmania .…”

“…In contrast, abnormally long telomeres were observed in the Ku knockout Drosophila cells ( 54 ). Notably, it has been recently reported that Leishmania mexicana telomeres were elongated from 250 to 850 bp to 250 to 3,000 bp upon deletion of the Ku70 or Ku 80 genes, and the lengths of the elongated telomeres then remain stable (unchanged) in the Ku-deficient L. mexicana cells ( 45 ).…”

“…To determine whether Ku is also involved in telomere maintenance in L. donovani , we compared the telomeres’ lengths in WT and Ku-deficient L. donovani cells by terminal restriction fragment (TRF) Southern blot and semiquantitative PCR analysis as described in methods ( 43 – 45 , 55 ). L. donovani promastigotes were harvested from culture every 3 weeks for total 9 weeks.…”

“…For example, it has been recently reported that Ku70/80 heterodimer suppresses MMEJ in Ligase IV-deficient progenitor B cells, deletion of Ku70 in this B cell line significantly increases MMEJ ( 33 ). In addition, Ku has also been shown to be involved in other cellular processes such as telomere maintenance, DNA replication, transcription, and translation control ( 7 , 8 , 37 – 45 ). In this study, to investigate whether Leishmania Ku70 and Ku80 proteins actively participate in DSB repair in the absence of key NHEJ factors Ligase IV and XRCC4, we generated Leishmania donovani Ku70 null mutants and Ku70/80 double gene null mutants.…”