Exo1 and Rad24 Differentially Regulate Generation of ssDNA at Telomeres of Saccharomyces cerevisiae cdc13-1 Mutants

Abstract

Cell cycle arrest in response to DNA damage depends upon coordinated interactions between DNA repair and checkpoint pathways. Here we examine the role of DNA repair and checkpoint genes in responding to unprotected telomeres in budding yeast cdc13-1 mutants. We show that Exo1 is unique among the repair genes tested because like Rad9 and Rad24 checkpoint proteins, Exo1 inhibits the growth of cdc13-1 mutants at the semipermissive temperatures. In contrast Mre11, Rad50, Xrs2, and Rad27 contribute to the vitality of cdc13-1 strains grown at permissive temperatures, while Din7, Msh2, Nuc1, Rad2, Rad52, and Yen1 show no effect. Exo1 is not required for cell cycle arrest of cdc13-1 mutants at 36Њ but is required to maintain arrest. Exo1 affects but is not essential for the production of ssDNA in subtelomeric YЈ repeats of cdc13-1 mutants. However, Exo1 is critical for generating ssDNA in subtelomeric X repeats and internal singlecopy sequences. Surprisingly, and in contrast to Rad24, Exo1 is not essential to generate ssDNA in X or single-copy sequences in cdc13-1 rad9⌬ mutants. We conclude that Rad24 and Exo1 regulate nucleases with different properties at uncapped telomeres and propose a model to explain our findings.

A Genomewide Suppressor and Enhancer Analysis of cdc13-1 Reveals Varied Cellular Processes Influencing Telomere Capping in Saccharomyces cerevisiae

“…For example, cdc13-1 was the primary tool used to show that Mec1, Mec3, Rad53 (Mec2), Rad17, and Rad24 are involved in DNA damage checkpoint control (Weinert et al 1994). Similarly, deletion of the EXO1 gene, which encodes a 59-39 exonuclease, allows cdc13-1 mutant cells to grow and divide at 27°, thus efficiently suppressing the temperaturesensitive telomere-capping defect (Maringele and Lydall 2002;Zubko et al 2004). This led to the discovery that Exo1 resects the ends of unprotected telomeres in at least two different situations (cdc13-1 mutants and yku70D cells), resulting in long stretches of ssDNA, which, in turn, act as a potent DNA damage signal (Maringele and Lydall 2002;Zubko et al 2004).…”

“…Similarly, deletion of the EXO1 gene, which encodes a 59-39 exonuclease, allows cdc13-1 mutant cells to grow and divide at 27°, thus efficiently suppressing the temperaturesensitive telomere-capping defect (Maringele and Lydall 2002;Zubko et al 2004). This led to the discovery that Exo1 resects the ends of unprotected telomeres in at least two different situations (cdc13-1 mutants and yku70D cells), resulting in long stretches of ssDNA, which, in turn, act as a potent DNA damage signal (Maringele and Lydall 2002;Zubko et al 2004). Importantly, the role of EXO1 and checkpoint genes in responding to uncapped telomeres appears to be conserved in mammals because it has been shown that deletion of exonuclease-1 or the CDK inhibitor p21 leads to an extension of life span in a mouse telomerase knockout model (Choudhury et al 2007;Schaetzlein et al 2007).…”

“…Importantly, the role of EXO1 and checkpoint genes in responding to uncapped telomeres appears to be conserved in mammals because it has been shown that deletion of exonuclease-1 or the CDK inhibitor p21 leads to an extension of life span in a mouse telomerase knockout model (Choudhury et al 2007;Schaetzlein et al 2007). Therefore the identification of new genetic interactions similar to those between RAD9 or EXO1 and cdc13-1 in yeast has the potential to identify novel conserved molecular pathways involved in the response to telomere uncapping, for example (Maringele and Lydall 2002;Zubko et al 2004).…”

Telomerase core components protect Candida telomeres from aberrant overhang accumulation

“…Notably, the overhang increase is reminiscent of the phenotypes of Saccharomyces cerevisiae mutants suffering from dysfunction of telomere binding proteins such as Cdc13p, Ku70, and Ku80 (14)(15)(16)(17). Analysis of such mutants indicated that loss of telomere protection engenders preferential degradation of the telomere C-strand, leading to G-strand overhang accumulation (18)(19)(20). Thus the phenotypes of the est2-⌬⌬ mutant suggest that, in C. albicans, the TERT/EST2 component may mediate a physically protective function akin to S. cerevisiae telomere binding proteins.…”

Bystander effect in human lymphocytes incubated with irradiated mitochondrial DNA deficient yeast cells

“…Haploid DLY640 strain S. cerevisiae (W303 background [11]) was kindly provided by Dr Mikhajlo K. Zubko (University of Newcastle, United Kingdom) and has genotype MATa ade2-1 trp1-1 S556 RADIOPROTECTION can1-100 leu2-3,112 his3-11, 15 ura3 GAL+ [psi+] ssd1-d2 RAD5. The rho 0 derivative strain was obtained by cultivation of the rho + cells in 2 ml of the liquid YPD medium containing 10 g/ml of ethidium bromide at the 28 • C without hesitation.…”