Telomerase-Null Survivor Screening Identifies Novel Telomere Recombination Regulators

Hong-Bo Tang,Xia-Jing Tong,Ning-Ning Liu,Jing Peng,Yan Hu,Jin-Qiu Zhou,Yang Zhang,Fei-Long Meng,Xiao-Hong Fu,Yi-Min Duan,Wei Dang,Michael J Mceachern

doi:10.1371/journal.pgen.1003208

Abstract

Telomeres are protein–DNA structures found at the ends of linear chromosomes and are crucial for genome integrity. Telomeric DNA length is primarily maintained by the enzyme telomerase. Cells lacking telomerase will undergo senescence when telomeres become critically short. In Saccharomyces cerevisiae, a very small percentage of cells lacking telomerase can remain viable by lengthening telomeres via two distinct homologous recombination pathways. These “survivor” cells are classified as either Type I or Type II, with each class of survivor possessing distinct telomeric DNA structures and genetic requirements. To elucidate the regulatory pathways contributing to survivor generation, we knocked out the telomerase RNA gene TLC1 in 280 telomere-length-maintenance (TLM) gene mutants and examined telomere structures in post-senescent survivors. We uncovered new functional roles for 10 genes that affect the emerging ratio of Type I versus Type II survivors and 22 genes that are required for Type II survivor generation. We further verified that Pif1 helicase was required for Type I recombination and that the INO80 chromatin remodeling complex greatly affected the emerging frequency of Type I survivors. Finally, we found the Rad6-mediated ubiquitination pathway and the KEOPS complex were required for Type II recombination. Our data provide an independent line of evidence supporting the idea that these genes play important roles in telomere dynamics.

Highlights

Telomeres are special DNA-protein structures found at the ends of eukaryotic chromosomes
By examining the telomere structures in 280 mutants, each of which lacks both a telomere-length-maintenance gene and telomerase RNA gene, we identified 32 genes that were not previously known to be involved in telomere recombination
Genomic DNA was extracted from each survival isolate, digested with the XhoI restriction enzyme, and analyzed by Southern blot with a TG probe to determine if the cells were Type I or Type II survivors (Figure 1A)

Summary

Introduction

Telomeres are special DNA-protein structures found at the ends of eukaryotic chromosomes. In budding yeast Saccharomyces cerevisiae, telomeric DNA consists of ,350 base pairs (bp) of TG1–3/C1–3 A repeats with a terminal single-stranded TG1–3 tract called a G-overhang [2]. Telomeric DNA can be maintained by either telomerase-mediated elongation or homologous recombination [3,4,5]. Telomerase is a highly specialized reverse transcriptase that adds telomeric DNA sequences to the 39 G-overhang using its intrinsic RNA template [3]. The telomerase pathway supercedes the recombination pathway as the predominant mechanism of telomeric DNA elongation [8,9]. In telomerase-null cells, telomeric DNA is maintained via a recombination pathway termed ‘‘alternative lengthening of telomeres’’ (ALT) [10]. 85% of immortalized human tumor cells use telomerase to maintain telomeres while 15% apply the ALT mechanism to maintain telomeres [11]

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Conclusion