Highlights d GLOE-Seq detects 3 0 -OH ends with nucleotide resolution in purified genomic DNA d GLOE-Seq maps single-strand breaks, lesions, and replication and repair intermediates d GLOE-Seq reveals insight into the use of ligases 1 and 3 in human cells d GLOE-Seq detects asymmetries in spontaneous nicks in yeast and human chromatin
Highlights d RPA foci induced by MMS or UV irradiation arise in postreplicative repair territories d Translesion synthesis and homologous recombination mediate resolution of RPA foci d Replication stress induced by damaged templates is resolved behind replication forks d Spontaneous replication problems are solved largely behind replication forks as well
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. Deletion of nts1 causes increased sensitivity to genotoxins and deregulated expression of Tf2 elements, long noncoding RNA, and subtelomeric and stress-related genes. Similar, but more pervasive, phenotypes are observed upon Clr6 inactivation, supporting the designation of complex I== as a mediator of a key subset of Clr6 functions. We also reveal that with the exception of Tf2 elements, the genome-wide loading sites and loci regulated by Clr6 I؆ do not correlate. Instead, Nts1 loads at genes that are expressed in midmeiosis, following oxidative stress, or are periodically expressed. Collective data suggest that Clr6 I== has (i) indirect effects on gene expression, conceivably by mediating higher-order chromatin organization of subtelomeres and Tf2 elements, and (ii) direct effects on the transcription of specific genes in response to certain cellular or environmental stimuli.
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 de-repress. The urg1 promoter also shows a large dynamic range and can be induced quickly (<2 h), but its implementation requires laborious strain construction and it cannot be used to study meiosis. To overcome these limitations we constructed a tetracycline-regulated system for inducible expression of chromosomal genes in fission yeast, which is easily established and implemented. In this system the promoter of a gene is replaced by simple one-step substitution techniques with a tetracycline-regulated promoter cassette (tetO7-TATACYC1) in cells where TetR/TetR′-based transcription activators/repressors are also produced. Using top1 and nse6 as reporter genes, we show that Top1 and Nse6 appear after just 30 min of activating tetO7-TATACYC1 and plateau after ~4–6 h. The amount of synthesised protein is comparable to that produced from the attenuated nmt1 promoter Pnmt8, which should be closer to wild type levels for most genes than those generated from excessively strong promoters and can be controlled by changing the concentration of the effector antibiotic. This system also works efficiently during meiosis, thus making it a useful addition to the toolkit of the fission yeast community.
Like in most other areas of cellular metabolism, the functions of the ubiquitin-like modifier SUMO in the maintenance of genome stability are manifold and varied. Perturbations of global sumoylation causes a wide spectrum of phenotypes associated with defects in DNA maintenance, such as hypersensitivity to DNA-damaging agents, gross chromosomal rearrangements and loss of entire chromosomes. Consistent with these observations, many key factors involved in various DNA repair pathways have been identified as SUMO substrates. However, establishing a functional connection between a given SUMO target, the cognate SUMO ligase and a relevant phenotype has remained a challenge, mainly because of the difficulties involved in identifying important modification sites and downstream effectors that specifically recognize the target in its sumoylated state. This review will give an overview over the major pathways of DNA repair and genome maintenance influenced by the SUMO system and discuss selected examples of SUMO's actions in these pathways where the biological consequences of the modification have been elucidated.
HighlightsWe examine the sumoylation of PARP-1 in response to different DNA ligands.We characterise the dynamics of the PARP-1 SUMO conjugate by NMR.Sumoylation of PARP-1 is stimulated by binding to intact, but not to damaged DNA.Sumoylation does not interfere with PARP-1's DNA binding or catalytic activity.PARP-1 sumoylation and catalytic activation follow independent regulatory mechanisms.
The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.
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