Either Rap1 or Cdc13 can protect telomeric single-stranded 3\u2032 overhangs from degradation in vitro

Rikard Runnberg,Saishyam Narayanan,Humberto Itriago,Marita Cohn

doi:10.1038/s41598-019-55482-3

Abstract

Telomeres, the DNA-protein structures capping the ends of linear chromosomes, are important for regulating replicative senescence and maintaining genome stability. Telomeres consist of G-rich repetitive sequences that end in a G-rich single-stranded (ss) 3′ overhang, which is vital for telomere function. It is largely unknown how the 3′ overhang is protected against exonucleases. In budding yeast, double-stranded (ds) telomeric DNA is bound by Rap1, while ssDNA is bound by Cdc13. Here, we developed an in vitro DNA 3′end protection assay to gain mechanistic insight into how Naumovozyma castellii Cdc13 and Rap1 may protect against 3′ exonucleolytic degradation by Exonuclease T. Our results show that Cdc13 protects the 3′ overhang at least 5 nucleotides (nt) beyond its binding site, when bound directly adjacent to the ds-ss junction. Rap1 protects 1–2 nt of the 3′ overhang when bound to dsDNA adjacent to the ds-ss junction. Remarkably, when Rap1 is bound across the ds-ss junction, the protection of the 3′ overhang is extended to 6 nt. This shows that binding by either Cdc13 or Rap1 can protect telomeric overhangs from 3′ exonucleolytic degradation, and suggests a new important role for Rap1 in protecting short overhangs under circumstances when Cdc13 cannot bind the telomere.

Highlights

Telomeres are specialized nucleoprotein structures localized at the ends of linear chromosomes that protect and stabilize the genome
In the work presented here, we have evaluated the ability of Cdc[13] and Rap[1] proteins to protect the telomeric 3′ overhang from 3′ exonucleolytic degradation through the use of an in vitro assay that utilizes recombinant N. castellii Cdc[13] and Rap[1], synthetic telomere mimicking DNA substrates and E. coli Exonuclease T
The oligonucleotides used in the 3′ DNA End Protection Assay (3′DEPA) contain the N. castellii telomeric repeat sequence and a short non-telomeric guide sequence

Summary

Introduction

Telomeres are specialized nucleoprotein structures localized at the ends of linear chromosomes that protect and stabilize the genome. In human cell lines without telomerase activity, telomere 3′ overhang length is proportional to the rate of telomere shortening per cell division[15]. T-loop formation involves the fold back and invasion of the 3′ overhang into the telomeric double-stranded region of the same chromosomal end, providing protection by effectively sequestering the 3′ overhang[18]. The length of human telomeric 3′ overhangs ranges from 12 up to several hundred nt[20,21,22] and at least 6 nt was shown to be required for the formation of a t-loop structure in vitro[23]. A large number of 3′ exonucleases are present in eukaryotic cells[29], yet few studies have addressed how they might act at the telomere 3′ overhang. The commercially available 3′ exonuclease ExoT is suitable for an in vitro model of telomere 3′ end protection

Methods

Results

Conclusion