SEGUROS

The number of expected Cdc13 bypass strains (nmd2 exo1 cdc13; nmd2 rad24 cdc13; nmd2 exo1 rad24 cdc13) was below what was expected when diploids DDY567 and 568 were grown in YPD (0% ethanol) (see columns indicated by red arrows in Figure 33). However, it appeared that the number of nmd2 exo1 cdc13 and nmd2 rad24 cdc13 strains was increasing with the percentage of ethanol in the media (compare these genotypes for 0% (red arrow) and 1% (green arrow) in Figure 33 and for 0% (red arrow) and 3% (green arrow) in Figure 34). Since ethanol increases telomere length, I

hypothesised that diploids with longer telomeres produced a higher frequency of Cdc13 bypass strains.

To test the hypothesis, genotype frequency was expressed as a percentage for each condition (i.e. 0% ethanol, 1% ethanol, 3% ethanol; 50 population

doublings) (Figure 35). It was found that the percentage of nmd2 exo1 cdc13 strains obtained dramatically increased, relative to 0% ethanol, when grown in 1% and 3% ethanol (error bars for 0%, 1% and 3% did not overlap). The percentage of nmd2 rad24 cdc13 strains also increased, compared to growth in 0% ethanol, after exposure to 3% ethanol (error bars for 0% and 3% did not overlap). Furthermore, a diploid made from a rif1 x nmd2 exo1 cdc13 cross, which has long telomeres initially, produced similarly high frequency of nmd2 exo1 cdc13 spores (18%; Figure 20). Interestingly, there was no change in the frequency of nmd2 exo1 rad24 cdc13 strains between 0% to 1% and 3% ethanol, perhaps because these have higher spore viability.

It appears that longer diploid telomeres lead to an increased frequency of nmd2 exo1 cdc13 and nmd2 rad24 cdc13 strains. It is possible that there is a higher degree of spore inviability for nmd2 exo1 cdc13 and nmd2 rad24 cdc13 genotypes when grown in YPD (0% ethanol), perhaps

because of short telomeres. Indeed, absence of nmd2 leads to short

telomeres, so perhaps this reduces spore viability in Cdc13 bypass strains. When diploids have longer telomeres this could increase the telomere length of the haploid progeny, allowing more Cdc13 bypass strains to form viable

Figure 35. Percentage of Cdc13 bypass strains obtained after diploid growth in ethanol

% obtained is the percentage of the total number of haploid strains analysed (for each % ethanol condition after 50 population doublings (PDs)), using the data shown in Figure 33 and Figure 34. The bars show the average from two independent diploid strains (sporulated to produce the haploids), the upper and lower bounds of the error bars show the individual value for each strain.

6.6 Discussion

The data in this chapter has shown that diploids with long, heterogeneous telomeres can generate cdc13 genotypes that are not normally viable. Such diploids can be produced when crossing a Cdc13 bypass strain with another haploid. In addition, diploids with slightly elongated telomeres (after growth in ethanol) also produce cdc13 genotypes that are not normally viable when telomeres are of wild-type length. Also, the frequency of nmd2 exo1 cdc13 and nmd2 rad24 cdc13 strains, previously identified as Cdc13 bypass strains, increased with telomere length. This indicates that longer telomeres increase the spore viability of these genotypes, perhaps because deletion of NMD2 leads to short telomeres which could lead to cell cycle arrest in strains lacking Cdc13. Since longer Y’ and X telomeres occurs by extension of TG1-3

repeats, it is possible that long tracts of these repeats promote survival without Cdc13. This hypothesis is support by the fact that cdc13 cells are inviable without Rad52, which promotes Type II recombination in which TG1-3 repeats

are amplified.

Long telomeres do not wholly explain Cdc13 bypass since an nmd2 exo1 cdc13 strain crossed with a rif1 strain had long telomeres but did not produce any cdc13 genotypes that are not normally viable. Nonetheless, the telomeres of this diploid recovered with passage, resembling a wild-type diploid telomere more closely after three passages. Therefore, perhaps Type II recombination is partially inhibited in the diploids heterozygous for NMD2, EXO1, CDC13 and RIF1 deletions but is not inhibited in diploids heterozygous for NMD2, RAD24, CDC13 and RIF1 deletions. Moreover, strains with the longest telomeres

produced fewer cdc13 genotypes (that are not normally viable) than those with slightly shorter telomeres.

Further work needs to be conducted to identify the other factors contributing to the growth of cdc13 genotypes that are not normally viable. One possible explanation for such genotypes is aneuploidy, when a strain has an extra chromosome carrying CDC13 as well as the deletion. To determine whether this is the case, PCR of the CDC13 gene in these strains could be carried out.

It would furthermore be informative to examine the telomere structure of haploid strains from diploids grown in ethanol compared to YPD. This would show whether telomere rearrangements and length are inherited by haploids. If so, this would provide further evidence that long telomeres promote Cdc13 bypass.

Long telomeres have been thought to be beneficial to health, since telomeres shorten with age and short telomeres may lead to genome instability and cancer (Lapham et al., 2015; Needham et al., 2015). Indeed, companies have been set up to offer telomere length assessment for individuals, for use as a biomarker for ageing and health. This is because shorter telomeres have been associated with alcohol consumption, smoking and obesity and it has been reported that healthy lifestyle changes increase telomere length in immune cells in individuals with cancer (Strandberg et al., 2012; Ornish et al., 2013; Joshu et al., 2015; Verde et al., 2015).

However, this chapter has provided evidence that long, heterogeneous telomeres contribute to cells surviving without the essential telomere-capping protein Cdc13. Furthermore, ethanol-lengthened telomeres in yeast lead to cdc13 genotypes not normally obtained and increases the frequency of Cdc13 bypass strains. Cell division in the absence of an essential telomere-capping protein is not necessarily beneficial, since the telomere end may have greater exposure to the DDR, which ultimately leads to genome instability. On the other hand, one cause of ageing is telomere shortening. Absence of a telomere capping protein may allow an ageing cell to maintain its viability by allowing telomeres to be extended and maintained by recombination. The disadvantage of telomere maintenance by recombination is that this can increase the risk of cancer, for example ALT cancer cells maintain telomeres by recombination.

Recent research has cast doubt on whether long telomeres are always

beneficial to health. Shorter telomeres are not necessarily associated with an increased cancer risk, although they do reduce cancer survival rates (Weischer et al., 2013). Also, it has been reported that subjects with a genetic

predisposition to short telomeres (those with SNPs associated with short leukocyte telomere length, e.g. SNPs in OBFC1) have lower cancer mortality, therefore those with genetically long telomeres have higher cancer mortality (Rode et al., 2015).

Interestingly, SNPs in OBFC1 (also known as human STN1) are associated with short telomeres but subjects do not have increased cancer mortality (Rode et al., 2015). This suggests that maximally functional (or indeed over-active) human STN1 may actually be harmful, leading to telomere maintenance of cells that should senesce or die due to their age or accumulated mutations (Rode et al., 2015). This appears to be the case in budding yeast, where increased Stn1 and Ten1 activity can allow Cdc13 bypass to occur, if the DDR is attenuated (Holstein et al., 2014). Long telomeres appear to contribute to bypass of Cdc13 without any other gene deletions (shown in this chapter), and it has been further shown that cells resembling Type I and Type II survivors, with substantial

telomere rearrangements, lose Cdc13 and maintain viability (Larrivée and Wellinger, 2006). It is possible that Stn1 and Ten1 provide sufficient telomere protection when telomeres are long, such that cells can survive without Cdc13.