Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres.

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs).

Recruitment, loading, and activation of the Smc5-Smc6 SUMO ligase.

Duplication of the genome poses one of the most significant threats to genetic integrity, cellular fitness, and organismal health. Therefore, numerous mechanisms have evolved that maintain replication fork stability in the face of DNA damage and allow faithful genome duplication. The fission yeast BRCT-domain-containing protein Brc1, and its budding yeast orthologue Rtt107, has emerged as a "hub" factor that integrates multiple replication fork protection mechanisms.

SUMO-targeted ubiquitin ligase activity can either suppress or promote genome instability, depending on the nature of the DNA lesion.

The posttranslational modifiers SUMO and ubiquitin critically regulate the DNA damage response (DDR). Important crosstalk between these modifiers at DNA lesions is mediated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitinates SUMO chains to generate SUMO-ubiquitin hybrids. These SUMO-ubiquitin hybrids attract DDR proteins able to bind both modifiers, and/or are degraded at the proteasome.

Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from .

The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site.

Large-Scale Purification of Small Ubiquitin-Like Modifier (SUMO)-Modified Proteins from .

Covalent protein modification by sumoylation (i.e., addition of small ubiquitin-like modifiers [SUMOs]) regulates a broad spectrum of critical functions in eukaryotic cells; however, usually ≤1% of a given protein is modified as a result of the highly dynamic nature of sumoylation.

Cooperativity of the SUMO and Ubiquitin Pathways in Genome Stability.

Covalent attachment of ubiquitin (Ub) or SUMO to DNA repair proteins plays critical roles in maintaining genome stability. These structurally related polypeptides can be viewed as distinct road signs, with each being read by specific protein interaction motifs. Therefore, via their interactions with selective readers in the proteome, ubiquitin and SUMO can elicit distinct cellular responses, such as directing DNA lesions into different repair pathways.

A novel histone deacetylase complex in the control of transcription and genome stability.

The acetylation state of histones, controlled by histone acetyltransferases (HATs) and deacetylases (HDACs), profoundly affects DNA transcription and repair by modulating chromatin accessibility to the cellular machinery. The Schizosaccharomyces pombe HDAC Clr6 (human HDAC1) binds to different sets of proteins that define functionally distinct complexes: I, I', and II. Here, we determine the composition, architecture, and functions of a new Clr6 HDAC complex, I'', delineated by the novel proteins Nts1, Mug165, and Png3.

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response.

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO.

A new versatile system for rapid control of gene expression in the fission yeast Schizosaccharomyces pombe.

The ability to regulate the expression of a gene greatly aids the process of uncovering its functions. The fission yeast Schizosaccharomyces pombe has so far lacked a system for rapidly controlling the expression of chromosomal genes, hindering its full potential as a model organism. Although the widely used nmt1 promoter displays a wide dynamic range of activity, it takes > 14-15 h to derepress.

Meiotic DNA joint molecule resolution depends on Nse5-Nse6 of the Smc5-Smc6 holocomplex.

Faithful chromosome segregation in meiosis is crucial to form viable, healthy offspring and in most species, it requires programmed recombination between homologous chromosomes. In fission yeast, meiotic recombination is initiated by Rec12 (Spo11 homolog) and generates single Holliday junction (HJ) intermediates, which are resolved by the Mus81-Eme1 endonuclease to generate crossovers and thereby allow proper chromosome segregation. Although Mus81 contains the active site for HJ resolution, the regulation of Mus81-Eme1 is unclear.

Dual recruitment of Cdc48 (p97)-Ufd1-Npl4 ubiquitin-selective segregase by small ubiquitin-like modifier protein (SUMO) and ubiquitin in SUMO-targeted ubiquitin ligase-mediated genome stability functions.

Protein modification by SUMO and ubiquitin critically impacts genome stability via effectors that "read" their signals using SUMO interaction motifs or ubiquitin binding domains, respectively. A novel mixed SUMO and ubiquitin signal is generated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitylates SUMO conjugates. Herein, we determine that the "ubiquitin-selective" segregase Cdc48-Ufd1-Npl4 also binds SUMO via a SUMO interaction motif in Ufd1 and can thus act as a selective receptor for STUbL targets.

The RecQ4 orthologue Hrq1 is critical for DNA interstrand cross-link repair and genome stability in fission yeast.

Of the five human RecQ family helicases, RecQ4, BLM, and WRN suppress distinct genome instability-linked diseases with severe phenotypes, often with indeterminate etiologies. Here, we functionally define Hrq1, a novel orthologue of RecQ4 from fission yeast. Biochemical analysis of Hrq1 reveals a DEAH box- and ATP-dependent 3'-5' helicase activity on various DNA substrates, including bubbles but not blunt duplexes, characteristic of the RecQ family.

DNA repair and global sumoylation are regulated by distinct Ubc9 noncovalent complexes.

Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2).

SUMO-targeted ubiquitin ligase, Rad60, and Nse2 SUMO ligase suppress spontaneous Top1-mediated DNA damage and genome instability.

Through as yet undefined proteins and pathways, the SUMO-targeted ubiquitin ligase (STUbL) suppresses genomic instability by ubiquitinating SUMO conjugated proteins and driving their proteasomal destruction. Here, we identify a critical function for fission yeast STUbL in suppressing spontaneous and chemically induced topoisomerase I (Top1)-mediated DNA damage. Strikingly, cells with reduced STUbL activity are dependent on tyrosyl-DNA phosphodiesterase 1 (Tdp1).

The dynamics and mechanism of SUMO chain deconjugation by SUMO-specific proteases.

SUMOylation of proteins is a cyclic process that requires both conjugation and deconjugation of SUMO moieties. Besides modification by a single SUMO, SUMO chains have also been observed, yet the dynamics of SUMO conjugation/deconjugation remain poorly understood. Using a non-deconjugatable form of SUMO we demonstrate the underappreciated existence of SUMO chains in vivo, we highlight the importance of SUMO deconjugation, and we demonstrate the highly dynamic nature of the SUMO system.

Nse1 RING-like domain supports functions of the Smc5-Smc6 holocomplex in genome stability.

The Smc5-Smc6 holocomplex plays essential but largely enigmatic roles in chromosome segregation, and facilitates DNA repair. The Smc5-Smc6 complex contains six conserved non-SMC subunits. One of these, Nse1, contains a RING-like motif that often confers ubiquitin E3 ligase activity.

A SIM-ultaneous role for SUMO and ubiquitin.

Ubiquitin and ubiquitin-like proteins (Ubls) share a beta-GRASP fold and have key roles in cellular growth and suppression of genome instability. Despite their common fold, SUMO and ubiquitin are classically portrayed as distinct, and they can have antagonistic roles. Recently, a new family of proteins, the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligases (STUbLs), which directly connect sumoylation and ubiquitylation, has been discovered.