A CDK-Dependent Phosphorylation of a Novel Domain of Rif1 Regulates its Function during Telomere Damage and Other Types of Stress.

Rif1 mediates telomere length, DNA replication, and DNA damage responses in budding yeast. Previous work identified several posttranslational modifications of Rif1, however none of these was shown to mediate the molecular or cellular responses to DNA damage, including telomere damage. We searched for such modifications using immunoblotting methods and the and models of telomere damage.

Telomere Dysfunction Triggers Palindrome Formation Independently of Double-Strand Break Repair Mechanisms.

Inverted chromosome duplications or palindromes are linked with genetic disorders and malignant transformation. They are considered by-products of DNA double-strand break (DSB) repair: the homologous recombination (HR) and the nonhomologous end joining (NHEJ). Palindromes near chromosome ends are often triggered by telomere losses.

Rif1 and Exo1 regulate the genomic instability following telomere losses.

Telomere attrition is linked to cancer, diabetes, cardiovascular disease and aging. This is because telomere losses trigger further genomic modifications, culminating with loss of cell function and malignant transformation. However, factors regulating the transition from cells with short telomeres, to cells with profoundly altered genomes, are little understood.

Polymerases ε and ∂ repair dysfunctional telomeres facilitated by salt.

Damaged DNA can be repaired by removal and re-synthesis of up to 30 nucleotides during base or nucleotide excision repair. An important question is what happens when many more nucleotides are removed, resulting in long single-stranded DNA (ssDNA) lesions. Such lesions appear on chromosomes during telomere damage, double strand break repair or after the UV damage of stationary phase cells.

A novel checkpoint and RPA inhibitory pathway regulated by Rif1.

Cells accumulate single-stranded DNA (ssDNA) when telomere capping, DNA replication, or DNA repair is impeded. This accumulation leads to cell cycle arrest through activating the DNA-damage checkpoints involved in cancer protection. Hence, ssDNA accumulation could be an anti-cancer mechanism.

Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects.

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time.

Polymerase epsilon is required to maintain replicative senescence.

Replicative senescence is a permanent cell cycle arrest in response to extensive telomere shortening. To understand the mechanisms behind a permanent arrest, we screened for factors affecting replicative senescence in budding yeast lacking telomere elongation pathways. Intriguingly, we found that DNA polymerase epsilon (Pol ε) acts synergistically with Exo1 nuclease to maintain replicative senescence.

Telomere maintenance and survival in saccharomyces cerevisiae in the absence of telomerase and RAD52.

Telomeres are essential features of linear genomes that are crucial for chromosome stability. Telomeric DNA is usually replenished by telomerase. Deletion of genes encoding telomerase components leads to telomere attrition with each cycle of DNA replication, eventually causing cell senescence or death.

Detecting repair intermediates in vivo: effects of DNA damage response genes on single-stranded DNA accumulation at uncapped telomeres in budding yeast.

Single-stranded DNA (ssDNA) is an important intermediate in many DNA repair pathways. Here we describe protocols that permit the measurement of ssDNA that has arisen in the yeast genome in vivo, in response to telomere uncapping. Yeast strains defective in DNA damage response (DDR) genes can be used to infer the roles of the corresponding proteins in regulating ssDNA production and in responding to ssDNA.

A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator.

Telomere capping is the essential function of telomeres. To identify new genes involved in telomere capping, we carried out a genome-wide screen in Saccharomyces cerevisiae for suppressors of cdc13-1, an allele of the telomere-capping protein Cdc13. We report the identification of five novel suppressors, including the previously uncharacterized gene YML036W, which we name CGI121.

Pulsed-field gel electrophoresis of budding yeast chromosomes.

Pulsed-field gel electrophoresis (PFGE) can be used to separate the 16 budding yeast chromosomes on the basis of size. Here we describe a detailed, practical protocol that will allow a novice to perform informative PFGE experiments. We first describe the culture of yeast prior to analysis, along with details of embedding cells in agarose before removal of cell walls.

The PAL-mechanism of chromosome maintenance: causes and consequences.

It is generally accepted that cells with extensive, un-repaired DNA damage can escape cell cycle arrest only by disabling checkpoint pathways and they usually perish, after several divisions, presumably due to catastrophic events on their chromosomes. Our recently discovered PAL-mechanism opens a new perspective, that some eukaryotic cells with short chromosome ends (telomeres), usually detected as DNA damage, can escape permanent cell cycle arrest (senescence) under special conditions, despite having intact checkpoints and even immortalize, despite lacking telomerase or other telomere elongation mechanisms. Here we present the first evidence that telomerase-lacking, senescent cells generate DNA damage (single stranded DNA) at internal chromosomal regions, while the telomere proximal single stranded DNA appears to be either lost or repaired.

Telomerase- and recombination-independent immortalization of budding yeast.

It is generally assumed that there are only two ways to maintain the ends of chromosomes in yeast and mammalian nuclei: telomerase and recombination. Without telomerase and recombination, cells enter senescence, a state of permanent growth arrest. We found that the decisive role in preventing senescent budding yeast cells from dividing is played by the Exo1 nuclease.

EXO1 plays a role in generating type I and type II survivors in budding yeast.

Telomerase-defective budding yeast cells escape senescence by using homologous recombination to amplify telomeric or subtelomeric structures. Similarly, human cells that enter senescence can use homologous recombination for telomere maintenance, when telomerase cannot be activated. Although recombination proteins required to generate telomerase-independent survivors have been intensively studied, little is known about the nucleases that generate the substrates for recombination.

EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Delta mutants.

We have examined the role of checkpoint pathways in responding to a yku70Delta defect in budding yeast. We show that CHK1, MEC1, and RAD9 checkpoint genes are required for efficient cell cycle arrest of yku70Delta mutants cultured at 37 degrees C, whereas RAD17, RAD24, MEC3, DDC1, and DUN1 play insignificant roles. We establish that cell cycle arrest of yku70Delta mutants is associated with increasing levels of single-stranded DNA in subtelomeric Y' regions, and find that the mismatch repair-associated EXO1 gene is required for both ssDNA generation and cell cycle arrest of yku70Delta mutants.