Unlike most other chromosomal loci, eukaryotic telomeres have unique structures attributed to their repetitive DNA sequence and binding proteins 
. Linear chromosome ends can be recognized as DNA double-stranded breaks and are thus often subjected to repair by non-homologous-end-joining and homologous recombination. It is possible that telomerase-null senescing cells are able to escape the fate of death as telomeres undergo lengthening and repair via homologous recombination. The distinct DNA makeup of Type I and Type II recombinational telomeres allowed us to carry out a genetic screening to identify genes that affect telomere recombination in telomerase-null cells.
Our candidate approach for screening telomere recombination genes had a few shortcomings. In our screening we only covered the 280 known TLM genes, which make up only 5.6% of the ~5,000 non-essential genes in S. cerevisiae
. It would be ideal to cover all non-essential genes in our screen. However, such a study would be too massive to undertake since the screening procedures included knocking out TLC1
in every strain, two to three-weeks passaging cells until they reach senescence and Southern blot experiments for multiple survivors in each mutant (see and ). The candidate approach we chose therefore had a strong bias. As a result, we might have missed potential genes that do not affect telomere length, but play important roles in telomere recombination. Another challenge to our screening approach came from the nature of different growth rates of the various mutants. Although we used heterozygous diploid mutants to generate spores of tlc1
Δ double mutants (Table S1
), for quite a few mutants we were not able to distinguish between a defect in a survivor pathway and synthetic lethality (Table S1
). The third issue that we were not able to resolve was to distinguish between hypo-Type I recombination and hyper-Type II recombination. The decrease of Type I survivor frequency seen in the mutants, such as rpa14
Δ () could be caused by either inhibition of Type I recombination or promotion of Type II recombination. In some Type II survivors, the amplified Y'-elements were detected in Southern blot assays (, Figure S1
), suggesting that the increase of Type II survivor frequency in these mutants was a result of enhanced Type II recombination rather than inhibited Type I recombination. This model is supported by the observation that in the nine mutants shown in and Figure S1
, the emerging events of Type I survivors were significantly reduced, but were not entirely blocked. The fourth issue that we had not taken into consideration during our primary screening was the effect of the initial telomere length of each mutant on the recombination pathways. It was recently proposed that longer telomeres, like those observed in rif1
Δ and rif2
Δ mutants could influence the type of recombination pathway used at the telomere 
. Additionally, it was shown that the mre11-A470T tlc1
Δ mutant promotes telomere recombination and bypass senescence efficiently because the Type I recombination occurs before growth limitation 
. Therefore, it would have been more appropriate to perform all of our screening steps starting with TLC1
diploids to obtain tlc1
Δ single and tlc1
Δ double mutants following tetrad dissection. The fifth issue with our screening approach was that we assumed the TLM genes only affect Type I or Type II recombination. Surprisingly, the telomere structure in the yku
mutants might actually be different from that of a typical Type I or Type II survivor ( and ). Therefore, genes that influence pathway(s) of telomere recombination other than that of Type I or Type II might have been overlooked. The sixth issue with our screen was that we only identified ten novel genes affecting Type I survivor formation (). This number might be underrepresent the true total because our primary screening was carried out with a relatively stringent criteria and as such we may have overlooked some genes that have minor influences on the frequency Type I survivor emergence.
Although our screening approach had some imperfections, we successfully identified thirty-two TLM genes that influence telomere recombination when overcoming senescence. Ten of these TLM genes affected the emerging frequency of Type I survivors while twenty-two were required for Type II survivor generation. A large portion of 280 TLM genes have not previously been characterized for their roles in telomere function other than the length of the telomeres in these deletion strains was altered. The positive results of our screen provide more direct evidence supporting the idea that some of these uncharacterized TLM genes do affect telomeres 
. Additionally, telomere recombination is a means by which cells repair defective telomeres and thus the genes involved in telomeric DNA recombination may also play a role in general DNA recombination/repair. Indeed, the TLM genes that affected either Type I or Type II recombination were also required for general DNA recombination (). The annotated functions of the thirty-two genes that we identified point to several pathways that might contribute to telomere maintenance ( and ). Some of the genes are known for functions like “rRNA processing,” “structural constituent of ribosome,” and “transport and membrane.” These gene products seem unlikely to play a direct role in telomere recombination. In contrast, the Pif1 helicase and the KEOPS complex are involved in “telomere capping and maintenance” 
and INO80 complex and Rad6 are associated with “chromatin remodeling and modification.” These genes are likely to play direct roles in telomere recombination.
The senescing pif1
Δ cells did not produce Type I survivors on solid medium () and the rad50
Δ triple mutant was not able to generate survivors in liquid medium (). These results indicated that Pif1 was required for Type I survivor generation. Interestingly, not all the rad51
Δ triple mutants were able to generate Type II survivors in liquid medium (). Therefore, we favor a model where Pif1 helicase is required for amplification of Y'-elements to form Type I survivors and promotes TG1–3
recombination to form Type II survivors (). Previous studies have shown that Pif1 takes part in mitochondrial DNA recombination 
, however, our data are the first to indicate that Pif1 is also involved in telomeric DNA recombination (). In the survivors of pif1
Δ mutants one group exhibited a severely delayed growth phenotype and had a unique telomere structure that differed from the characteristics of either Type I or Type II (). We speculate that these types of survivors require RAD50
to maintain telomeres since no survivors were recovered in the pif1
Δ triple mutant (). In the future it will be interesting to examine how the short telomeres are maintained in these survivors.
Chromatin remodeling complexes have been shown by others to play roles in DNA repair processes via homologous recombination 
. However, a causal link between chromatin structure alteration and recombination has not yet been well established. We found that in the absence of active chromatin remodeling by the INO80 complex, telomere Type I recombination was unable to efficiently take place (), suggesting that the alteration of chromatin structure is a pre-requisite to the Type I recombination process at telomeres. Our results could provide an explanation for the previous observation that ies3
Δ cells generated survivors later than the est1
Δ single mutant 
, as ies3
Δ cells likely have a lower efficiency of Type I survivor generation than est1
Δ cells. SAP30
, which encodes a subunit of histone deacetylase Rpd3 complex, was also identified in our screening. Deletion of SAP30
dramatically reduced the emerging rate of the Type I survivors (), suggesting that the Rpd3 histone deacetylase complex may also inhibit Type I recombination. The SWR1 complex is another chromatin remodeling complex that belongs to the INO80 family of remodeling enzymes. SWR1 and INO80 complexes share four common subunits: Rvb1, Rvb2, Arp4 and Act1 
. It will be intriguing to determine if other chromatin remodeling enzymes like SWR1 or histone modification enzymes play roles in telomere recombination.
The KEOPS complex gains its name from “Kinase, Endopeptidase and Other Proteins of small Size” 
, and is comprised of five small proteins (Bud32, Kae1, Pcc1, Gon7 and Cgi121) which form a stable complex in vitro
and in vivo
. Bud32 has kinase activity while Kae1 maintains endopeptidase activity 
. The KEOPS complex or its subunit(s) are involved in several biological processes, to which each KEOPS subunit seems to contribute unequally. Pcc1, Gon7, Kae1 and Bud32, for example, are recruited to several genomic loci and affect gene transcription 
. Kae1 contributes to faithful chromosome segregation 
while Bud32, Cgi121, Gon7 regulate cell polarity in bud-site selection 
. Additionally, Bud32, Kae1 and Pcc1 are essential for a universal tRNA modification called threonyl carbamoyl adenosine (t6A), for which Cgi121 is dispensable 
. Moreover, all the subunits of the KEOPS complex appear to play roles in telomere uncapping and telomere length regulation 
. Our screen elucidated a novel function of the KEOPS complex in telomere recombination, as deficiency of any subunit of the KEOPS complex led to the failure in generating Type II survivors in the tlc1
Δ mutant (). The molecular mechanism by which the KEOPS complex influences telomere recombination remains unclear. A previous study by Downey et al. showed that mutation of the KEOPS complex decreased the amount of single-stranded telomeric DNA in the cdc13-1
. It is possible that the KEOPS complex facilitates the formation of the telomeric 3′-overhang and promotes recombination of TG-tracts. Coincidently, SUA5
, a telomeric single-stranded DNA binding protein, is required for both Type II recombination and t6A modification of tRNA 
. It will be interesting to determine whether SUA5
is a downstream target of the KEOPS complex and if it functions in the same pathway in regulating telomere recombination. It is possible that Sua5 is a substrate of the Bud32 kinase.
In summary, our screen identified dozens of genes that regulate telomere recombination pathways. Because of the complexity of the recombination process, the molecular mechanisms of telomere recombination remain elusive. Our work not only provides important clues for beginning to understand how telomere recombination is coordinated, but also offers new insights into general DNA repair processes via homologous recombination.