Proteins that function at telomeres : genetic and biochemical investigation

Abstract

The telomere is a nucleoprotein structure found at the ends of all eukaryotic chromosomes that plays a key role in ageing and cellular proliferation. It prevents chromosome ends being recognised and processed as double strand breaks and therefore preserves genomic integrity. This study aimed to further investigate the role of a telomere capping protein complex found in S. cerevisiae, which comprises three proteins, Cdc13, Stn1 and Ten1 forming the CST complex. The CST complex associates with the single stranded G‐rich overhangs found at telomere ends acting as a nucleating centre for various telomere associated factors, and preventing access to enzymes that might process the telomere as a double strand break, in particular exonucleases. This is evidenced from genetic studies involving temperature‐sensitive mutants of CDC13, STN1, TEN1 all of which accumulated single stranded DNA at the telomere end resulting in activation of the G2/M checkpoint. However, the ability of these proteins to protect telomere‐like structures in vitro has yet to be demonstrated. Furthermore, similarities have been drawn between the domain architecture of the components of the CST complex and Replication protein A (RPA) a ubiquitous single stranded DNA binding protein extensively involved in DNA metabolism. This has led to the suggestion that CST could be a telomere specific version of RPA. In this study, an in vitro telomere protection assay was developed and optimised to directly test the ability of CST and RPA to inhibit 5’ to 3’ resection of telomere mimics and non‐telomere controls. It was found that while RPA was able to protect telomere and non‐telomere control substrates from resection, Cdc13 only protected telomeres. Furthermore, it was found that the DNA binding domain of Cdc13 was able to inhibit resection by the 5’ to 3’ exonuclease, λ‐exonuclease, and was able to outcompete RPA for binding to the 3’ G‐rich overhang found at the end of the telomere mimic. The two small subunits of the CST complex, Stn1 and Ten1, were not able to inhibit nuclease resection by λ‐exonuclease at telomere mimics or nontelomere controls. Two synthetic genetic arrays and quantitative fitness analyses were carried out using temperature‐sensitive alleles of STN1 and RPA3 (the second largest subunit of the CST ii complex and the smallest subunit of the RPA heterotrimer respectively). The aim of these screens was to determine the extent to which the genetic interaction profiles of these screens overlapped with that demonstrated previously for cdc13‐1. It was found that, similarly to cdc13‐1, the stn1‐13 temperature‐sensitive phenotype was suppressed by deletion of EXO1 or nonsense‐mediated mRNA decay genes. However, deletion of a genome integrity checkpoint protein required for cell cycle arrest in G2/M, RAD9, in the stn1‐13 background, enhanced the temperature‐sensitive phenotype, suggesting that the G2/M checkpoint was important for the vitality of stn1‐ 13 strains in contrast to cdc13‐1. It was also found that deletion of the two subunits of the Ku heterodimer (YKU70 and YKU80) did not affect the growth of rpa3‐313 strains in contrast to cdc13‐1 and stn1‐13 strains where these deletions had a negative effect on growth. In addition deletion of EXO1 had no effect on the fitness of rpa3‐313 strains in contrast to its suppressive effect on temperature‐sensitivity in the cdc13‐1 and stn1‐ 13 background. These results demonstrate biochemically that Cdc13 and RPA inhibit 5’ to 3’ resection at telomere ends. Furthermore they demonstrate the importance of STN1 in preserving the telomere end, and the involvement of the nonsense‐mediated mRNA decay pathway in disrupting CST‐mediated telomere capping. Finally they underline the difficulty of disentangling the role of RPA in telomere capping using genetic techniques due to its extensive involvement in DNA metabolism throughout the cell.