Telomere-binding proteins Taz1 and Rap1 regulate DSB repair and suppress gross chromosomal rearrangements in fission yeast

Abstract

Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs.

Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination

“…It has been shown that truncation ends are healed by de novo telomere addition [52][53][54]. To determine the chromosomal sites to which telomere sequences have been added, we recovered chromosomal truncations from agarose gels and amplified the breakpoints using a pair of ChL C and telomere primers: M13R-C19 or M13R-T1 [55]. DNA sequencing of the PCR products revealed that telomere sequences (G 2-5 TTAC (A) (C)) [56] were added either within or outside centromere repeats (Tables 1 and 2).…”

Nuclear genome organization in fungi: from gene folding to Rabl chromosomes

“…2 ) (Steinberg-Neifach and Lue 2015 , Erlendson et al 2017 , Xue et al 2017 ). In S. cerevisiae , RAP1 directly recognizes telomeric dsDNA and recruits RIF1/2 ( R AP1 I nteracting F actor 1/2 ) to regulate telomere length, while TBF1 ( T TAGGG B inding F actor 1 ), a yeast ortholog of human TRF1, binds to single stranded telomeric DNA to antagonize improper telomere lengthening (Bilaud et al 1996 , Li et al 2000 , Hediger et al 2006 , Pitt et al 2008 , Kabir et al 2010 , Kaizer et al 2015 , Irie et al 2019 ). In contrast, the S. pombe shelterin complex contains the proteins TAZ1 ( T elomere- A ssociated in Schi Z osaccharomyces pombe 1 ; a homolog of mammalian TRF1/2), RAP1, RIF1, POT1, TPZ1 (T elomeres P rotection protein in Schi Z osaccharomyces pombe 1 ), and CCQ1 ( C oiled- C oil Quantitatively enriched protein 1 ) for protecting telomeric DNA and silencing subtelomeric chromatin (Cooper et al 1997 , Baumann and Cech 2001 , Kanoh and Ishikawa 2001 , Miyoshi et al 2008 , Fujita et al 2012 , Deng et al 2015 , Zofall et al 2016 , Irie et al 2019 ).…”

Flexible Attachment and Detachment of Centromeres and Telomeres to and from Chromosomes

“…The GCR assay was recently established in S. pombe , which estimated the frequency of telomere healing in the wild-type context as ~2 × 10 −9 and demonstrated its evolutionary conservation as the primal GCR event [ 59 ] ( Table 1 ). The rate of GCRs per unit length of DNA was matched fairly well between S. cerevisiae (~3 × 10 −11 /kb) [ 109 ] and S. pombe (~1 × 10 −10 /kb) [ 59 ], suggesting a universal, DNA sequence-independent, and DNA length-dependent mechanism for the development of DSBs as a GCR trigger.…”

“…In the GCR assay in S. pombe , the telomere capping proteins Taz1 and Rap1 suppressed telomere healing [ 59 ], which is somewhat reminiscent of telomere healing regulation at the site-specific DSB in S. cerevisiae [ 104 , 106 ]. However, the regulation in S. cerevisiae was fully dependent on the telomeric repeat sequences near the DSB, whereas no preference for telomere repeat-like sequences in the DNA surrounding various breakpoints was found in the GCR assay in both S. cerevisiae and S. pombe [ 59 , 108 ]. Instead, direct involvement of S. pombe Taz1 and Rap1 proteins in the DSB repair pathway was proposed based on a different assay using site-specific endonuclease induction [ 59 ].…”