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

Abstract

Summary Homologous recombination and repair factors are known to promote both telomere replication and recombination‐based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance in Ustilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase‐positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase‐negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi‐solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II‐like as well as ALT‐like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I‐like telomere features. In addition, we observed direct physical interactions between Blm and two telomere‐binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination.

Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing

“…1B). The challenge becomes even more significant when there are multiple, overlapping TRF clusters, as was the case in the earlier studies 13 15 .…”

“…These previous studies, unlike the TRF analysis in Fig. 1B, were based on probing PstI-generated TRF clusters using a telomere repeat (TTAGGG) probe, and estimated the average telomere lengths to be 300-400 bp or 400 bp 13 15 . One of the studies also yielded a telomere size variation of ~ 300 bp 13 .…”

“…In contrast, even though some budding yeast helicase and repair proteins have also been implicated in telomere maintenance, this role is typically more discernable in telomerase-negative cells - e.g., Saccharomyces cerevisiae SGS1 or RAD51 deletion can accelerate senescence of telomerase-negative mutants, but does not cause telomere shortening in wild type cells 16 17 . By characterizing the U. maydis helicase/repair mutants and trt1 ∆ (telomerase-null) separately and in combination, we showed that the helicase/repair genes probably promote telomere maintenance through overlapping pathways that are distinct from the telomerase pathway 15 . For example, in contrast to the progressive shortening phenotype of trt1 ∆, telomeres are stably maintained in the helicase/repair mutants, and combining the helicase/repair mutations with trt1 ∆ triggers accelerated senescence and telomere loss.…”

“…We are particularly interested in understanding the interplay between telomere replication and telomerase in sustaining telomere length. For this purpose, we have been exploring the Basidiomycete fungus Ustilago maydis as a useful model system 13 14 15 . In comparison to the standard budding and fission yeast models, U. maydis has a number of advantages, especially in regard to the study of telomere replication.…”

Brca2 abrogation engages with the alternative lengthening of telomeres via break‐induced replication

“…We showed in Caenorhabditis elegans that depletion of BRC-2, an orthologue of Brca2, results in a delay of senescence, accompanied by telomere length elongation [13]. Similarly, abrogation of Brh2, an orthologue of Brca2 in fungus, Ustilago maydis, led to an ALT-like feature [14]. Here, we asked whether this function of Brca2 in suppressing ALT is conserved in mice.…”