Deletion of MEC1 suppresses the replicative senescence of the cdc13-2 mutant in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, telomerase recruitment to telomeres depends on a direct interaction between Cdc13, a protein that binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. The cdc13-2 allele disrupts telomerase association with telomeres, resulting in progressive telomere shortening and replicative senescence. The Mec1/ATR kinase is both a positive and a negative regulator of telomerase activity and is required for the cell cycle arrest in telomerase-deficient senescent cells. In this study, we find that the deletion of MEC1 suppresses the replicative senescence of cdc13-2. This suppression is dependent on telomerase, indicating that Mec1 antagonizes telomerase-mediated telomere extension in cdc13-2 cells to promote senescence.

High-throughput replica-pinning approach to screen for yeast genes controlling low-frequency events.

Saccharomyces cerevisiae is a leading model system for genome-wide screens, but low-frequency events (e.g., point mutations, recombination events) are difficult to detect with existing approaches. Here, we describe a high-throughput screening technique to detect low-frequency events using high-throughput replica pinning of high-density arrays of yeast colonies. This approach can be used to screen genes that control any process involving low-frequency events for which genetically selectable reporters are available, e.g., spontaneous mutations, recombination, and transcription errors. For complete details on the use and execution of this protocol, please refer to (Novarina et al., 2020a, 2020b).

Suppression of cdc13-2-associated senescence by pif1-m2 requires Ku-mediated telomerase recruitment.

In Saccharomyces cerevisiae, recruitment of telomerase to telomeres requires an interaction between Cdc13, which binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. A second pathway involving an interaction between the yKu complex and telomerase RNA (TLC1) contributes to telomerase recruitment but cannot sufficiently recruit telomerase on its own to prevent replicative senescence when the primary Cdc13-Est1 pathway is abolished-for example, in the cdc13-2 mutant. In this study, we find that mutation of PIF1, which encodes a helicase that inhibits telomerase, suppresses the replicative senescence of cdc13-2 by increasing reliance on the yKu-TLC1 pathway for telomerase recruitment. Our findings reveal new insight into telomerase-mediated telomere maintenance.

A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers.

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the Skp, Cullin, F-box containing (SCF) ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (Histone Deacetylase 4, HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work identified a novel potential drug target for tumors that overexpress MELK.

Two maize Kip-related proteins differentially interact with, inhibit and are phosphorylated by cyclin D-cyclin-dependent kinase complexes.

The family of maize Kip-related proteins (KRPs) has been studied and a nomenclature based on the relationship to rice KRP genes is proposed. Expression studies of KRP genes indicate that all are expressed at 24 h of seed germination but expression is differential in the different tissues of maize plantlets. Recombinant KRP1;1 and KRP4;2 proteins, members of different KRP classes, were used to study association to and inhibitory activity on different maize cyclin D (CycD)-cyclin-dependent kinase (CDK) complexes. Kinase activity in CycD2;2-CDK, CycD4;2-CDK, and CycD5;3-CDK complexes was inhibited by both KRPs; however, only KRP1;1 inhibited activity in the CycD6;1-CDK complex, not KRP4;2. Whereas KRP1;1 associated with either CycD2;2 or CycD6;1, and to cyclin-dependent kinase A (CDKA) recombinant proteins, forming ternary complexes, KRP4;2 bound CDKA and CycD2;2 but did not bind CycD6;1, establishing a differential association capacity. All CycD-CDK complexes included here phosphorylated both the retinoblastoma-related (RBR) protein and the two KRPs; interestingly, while KRP4;2 phosphorylated by the CycD2;2-CDK complex increased its inhibitory capacity, when phosphorylated by the CycD6;1-CDK complex the inhibitory capacity was reduced or eliminated. Evidence suggests that the phosphorylated residues in KRP4;2 may be different for every kinase, and this would influence its performance as a cyclin-CDK inhibitor.