Different types of gross chromosomal rearrangements (GCRs), including translocations, interstitial deletions, terminal deletions with de novo telomere additions, and chromosome fusions, are observed in many cancers. Multiple pathways, such as S-phase checkpoints, DNA replication, recombination, chromatin remodeling, and telomere maintenance that suppress GCRs have been identified. To experimentally expand our knowledge of other pathway(s) that suppress GCRs, we developed a generally applicable genome-wide screening method. In this screen, we identified 10 genes (ALO1, CDC50, CSM2, ELG1, ESC1, MMS4, RAD5, RAD18, TSA1, and UFO1) that encode proteins functioning in the suppression of GCRs. Moreover, the breakpoint junctions of GCRs from these GCR mutator mutants were determined with modified breakpoint-mapping methods. We also identified nine genes (AKR1, BFR1, HTZ1, IES6, NPL6, RPL13B, RPL27A, RPL35A, and SHU2) whose mutations generated growth defects with the pif1⌬ mutation. In addition, we found that some of these mutations changed the telomere size.

Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress. The Mec1/Rad53/Dun1 DNA damage response kinase cascade exhibits multifaceted controls over RNR activity including the regulation of the RNR inhibitor, Sml1. After DNA damage, Sml1 is degraded leading to the up-regulation of dNTP pools by RNR. Here, we probe the requirements for Sml1 degradation and identify several sites required for in vivo phosphorylation and degradation of Sml1 in response to DNA damage. Further, in a strain containing a mutation in Rnr1, rnr1-W688G, mutation of these sites in Sml1 causes lethality. Degradation of Sml1 is dependent on the 26S proteasome. We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4.

Telomeres, the ends of linear eukaryotic chromosomes, shorten due to incomplete DNA replication and nucleolytic degradation. Cells counteract this shortening by employing a specialized reverse transcriptase called telomerase, which uses deoxyribonucleoside triphosphates (dNTPs) to extend telomeres. Intracellular dNTP levels are tightly regulated, and perturbation of these levels is known to affect DNA synthesis. We examined whether altering the levels of the dNTP pools or changing the relative ratios of the four dNTPs in Saccharomyces cerevisiae would affect the length of the telomeres. Lowering dNTP levels leads to a modest shortening of telomeres, while increasing dNTP pools has no significant effect on telomere length. Strikingly, altering the ratio of the four dNTPs dramatically affects telomere length homeostasis, both positively and negatively. Specifically, we find that intracellular deoxyguanosine triphosphate (dGTP) levels positively correlate with both telomere length and telomerase nucleotide addition processivity in vivo. Our findings are consistent with in vitro data showing dGTP-dependent stimulation of telomerase activity in multiple organisms and suggest that telomerase activity is modulated in vivo by dGTP levels.A LL eukaryotes, as well as some prokaryotes with linear chromosomes, contain repetitive sequences called telomeres at the ends of their DNA. Telomeric DNA is bound by proteins that protect chromosome ends from being recognized as genotoxic DNA double-strand breaks in need of repair (Jain and Cooper 2010). However, telomeres shorten due to incomplete DNA replication and nucleolytic degradation. Left unchecked, this telomere erosion eventually results in very short, unprotected telomeres, leading to cell-cycle arrest and replicative senescence (Lundblad and Szostak 1989;Harley et al. 1990;Yu et al. 1990).Telomere shortening is counteracted by a specialized reverse transcriptase called telomerase (Greider and Blackburn 1985), whose core consists of a protein catalytic subunit and an RNA moiety-hTERT and hTR, respectively, in humans (Feng et al. 1995;Nakamura et al. 1997), and Est2 and TLC1, respectively, in the budding yeast Saccharomyces cerevisiae (Singer and Gottschling 1994;Lingner et al. 1997). Telomerase extends telomeres by repeated reverse transcription of a short sequence to the 39 ends of telomeres, using the RNA subunit as a template (Greider and Blackburn 1989;Yu et al. 1990;Singer and Gottschling 1994). Although the sequence of the telomeric repeats differs between species, a common feature is that they are all G-rich. In vertebrates, the repeat sequence is TTAGGG (Meyne et al. 1989), while in S. cerevisiae, the telomeric repeats have a consensus sequence of (TG) 0-6 TGGGTGTG(G) 0-1 (Forstemann and Lingner 2001).Deoxyribonucleoside triphosphates (dNTPs) are the building blocks of DNA, and their production needs to be tightly regulated as imbalances in dNTP pools can be mutagenic (Reichard 1988). In S. cerevisiae, the sole mode of dNTP production is through de novo dN...

Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase. Cin8's kinetochore localization depends on the Ndc80 complex, kinetochore microtubules, and the Dam1 complex. Consistent with its kinetochore localization, a Cin8 deletion induces a loss of tension at the Ndc80 microtubule binding domains and a major delay in mitotic progression. Cin8 associates with Protein Phosphatase 1 (PP1), and mutants that inhibit its PP1 binding also induce a loss of tension at the Ndc80 microtubule binding domains and delay mitotic progression. Taken together, our results suggest that Cin8-PP1 plays a critical role at kinetochores to promote accurate chromosome segregation by controlling Ndc80 attachment to microtubules.