Mre11 and Blm-Dependent Formation of ALT-Like Telomeres in Ku-Deficient Ustilago maydis

Abstract

A subset of human cancer cells uses a specialized, aberrant recombination pathway known as ALT to maintain telomeres, which in these cells are characterized by complex aberrations including length heterogeneity, high levels of unpaired C-strand, and accumulation of extra-chromosomal telomere repeats (ECTR). These phenotypes have not been recapitulated in any standard budding or fission yeast mutant. We found that eliminating Ku70 or Ku80 in the yeast-like fungus Ustilago maydis results initially in all the characteristic telomere aberrations of ALT cancer cells, including C-circles, a highly specific marker of ALT. Subsequently the ku mutants experience permanent G2 cell cycle arrest, accompanied by loss of telomere repeats from chromosome ends and even more drastic accumulation of very short ECTRs (vsECTRs). The deletion of atr1 or chk1 rescued the lethality of the ku mutant, and “trapped” the telomere aberrations in the early ALT-like stage. Telomere abnormalities are telomerase-independent, but dramatically suppressed by deletion of mre11 or blm, suggesting major roles for these factors in the induction of the ALT pathway. In contrast, removal of other DNA damage response and repair factors such as Rad51 has disparate effects on the ALT phenotypes, suggesting that these factors process ALT intermediates or products. Notably, the antagonism of Ku and Mre11 in the induction of ALT is reminiscent of their roles in DSB resection, in which Blm is also known to play a key role. We suggest that an aberrant resection reaction may constitute an early trigger for ALT telomeres, and that the outcomes of ALT are distinct from DSB because of the unique telomere nucleoprotein structure.

Contributions of recombination and repair proteins to telomere maintenance in telomerase‐positive and negative Ustilago maydis

“…RAD51 and BRCA2 have also been reported to enhance telomere replication, though the precise mechanisms are not understood (Badie et al ., ; Zimmer et al ., ). We showed previously that deleting U. maydis blm , rad51 and brh2 each caused substantial telomere loss (Yu et al ., 2013; 2015) (Fig. ).…”

“…First, just like the human KU complex (but unlike any other system that has been studied to date), the U. maydis Ku complex is essential for cell viability and the loss of Ku expression causes massive telomere aberrations (de Sena‐Tomas et al ., ; Yu et al ., ). Second, the two core recombination proteins in U. maydis (i.e., Rad51 and Brh2) and the Blm helicase are all required for normal telomere maintenance in telomerase‐positive cells (Badie et al ., ; Yu et al ., 2013; 2015); deleting each gene causes a significant reduction in telomere lengths in the mutants. Thus, the recombination repair proteins in U. maydis may play a role in promoting telomere replication, just like their human counterparts.…”

Telomere recombination pathways: tales of several unhappy marriages

“…It had been shown earlier that human KU86 (the human Ku80 gene) is essential and that KU86 -null cancer cells rapidly lose viability due to massive loss of telomere repeats from chromosome ends (Wang, et al 2009). Remarkably, these observations were recently shown to hold true for the U. maydis ku70 and ku80 genes (even though other vertebrate and mammalian KU mutants are known to be viable) (de Sena-Tomas, et al 2015, Yu, et al 2015). The non-viability of the Ustilago Ku70- and Ku80-deficient cells can be suppressed by atr1 or chk1 deletion, and these ku/checkpoint double mutants exhibit all the hallmark telomere aberrations of ALT cancer cells, including telomere length heterogeneity and high levels of ECTRs.…”

“…While these fungal mutants do not recapitulate all the telomere phenotypes of ALT cells, they have provided general insights on the variety of recombination factors and pathways that can be triggered at telomeres. More recently, a fungal model that exhibits greater similarity to ALT than previous models was reported in Ustilago maydis (de Sena-Tomas, et al 2015, Yu, et al 2015). U. maydis is a basidiomycete that is distantly related to budding and fission yeasts, and features of the U. maydis ALT model provide insights on the reasons that may underlie mechanistic distinctions and resemblances between different telomere recombination pathways in different organisms.…”

“…Because these factors were implicated in ALT based on co-localization or partial effects of knockdown, it remains unclear whether they are indispensable. An apparent discrepancy between ALT and the Ustilago model is in regard to the role of Top3: knockdown of human TOP3α inhibited proliferation of and reduced telomere stability in ALT cells (Temime-Smaali, et al 2008, Tsai, et al 2006), but deletion of top3 enhanced telomere length heterogeneity and C-circle formation in U. maydis (Yu, et al 2015). While this discrepancy may point to significant mechanistic differences between the model and ALT cancer cells, an alternative explanation should be considered.…”