Eukaryotic cells use multiple, highly conserved mechanisms to contend with ultraviolet-light-induced DNA damage. One important response mechanism is transcription-coupled repair (TCR), during which DNA lesions in the transcribed strand of an active gene are repaired much faster than in the genome overall. In mammalian cells, defective TCR gives rise to the severe human disorder Cockayne's syndrome (CS). The best-studied CS gene, CSB, codes for a Swi/Snf-like DNA-dependent ATPase, whose yeast homologue is called Rad26 (ref. 4). Here we identify a yeast protein, termed Def1, which forms a complex with Rad26 in chromatin. The phenotypes of cells lacking DEF1 are consistent with a role for this factor in the DNA damage response, but Def1 is not required for TCR. Rather, def1 cells are compromised for transcript elongation, and are unable to degrade RNA polymerase II (RNAPII) in response to DNA damage. Our data suggest that RNAPII stalled at a DNA lesion triggers a coordinated rescue mechanism that requires the Rad26-Def1 complex, and that Def1 enables ubiquitination and proteolysis of RNAPII when the lesion cannot be rapidly removed by Rad26-promoted DNA repair.

Transcription‐coupled repair (TCR) is a universal sub‐pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription‐blocking DNA lesions and permits rapid resumption of the vital process of transcription. A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS). Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B. Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26. A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function. In analogy with CS, preferential repair of UV‐induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired. Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV‐, cisPt‐ or X‐ray sensitivity, explaining why it was not isolated as a mutant before. Recovery of growth after UV exposure was somewhat delayed in rad26. These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity.

Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30%Yo of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and radl6 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD' cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and radl16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs.

Diacylglycerol lipase-a (DAGL-a) is an intracellular, multidomain protein responsible for the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system. [1] 2-AG is an endogenous signaling lipid that interacts with the cannabinoid CB1 and CB2 receptors. [2] Little is known about the regulation of its biosynthetic pathway and it is largely unclear to what extent 2-AG is responsible for distinct cannabinoid CB1 receptor mediated biological processes. Selective inhibitors of DAGL-a may contribute to a more fundamental understanding of the physiological role of 2-AG and may serve as potential drug candidates for the treatment of obesity and neurodegenerative diseases. [3] Currently, there are no selective inhibitors and activity-based probes available for the study of DAGL-a. [4] The identification of selective DAGL-a inhibitors is hampered by a lack of structural knowledge of the target, and a lack of assays that make use of endogenous DAGL-a activity in proteomes. No crystal structures are available and no homology models have been reported to aid hit identification and to guide optimization of the inhibitors. Determination of the selectivity of the inhibitors in native tissues is important because DAGL-a belongs to the serine hydrolase family, which contains more than 200 members with various physiological functions. [5] Fluorophosphonate (FP)based probes are routinely employed in competitive activitybased protein profiling (ABPP) experiments to determine the selectivity of serine hydrolase inhibitors in complex proteomes. DAGL-a, however, does not react with these activitybased probes. [6] Therefore, a new probe that can label native DAGL-a would be of value for studying the potency and selectivity of novel DAGL-a inhibitors in brain proteomes.Here we present a strategy that combines a knowledge-based in silico design approach and the development of a novel activity-based probe (ABP), based on the nonselective DAGL-a inhibitor tetrahydrolipstatin (THL; also known as Orlistat, a drug used for the treatment of obesity). This strategy resulted in the rapid identification of DAGL-a inhibitors with a new chemotype and high selectivity in the brain proteome.To identify novel DAGL-a inhibitors, we built a pharmacophore model based on THL using Discovery Studio Software Suite from Accelrys. Since THL can assume many different conformations, we searched the protein crystallographic database for crystal structures with a bioactive conformation for THL. A cocrystal structure of THL with fatty acid synthase (pdb-code: 2PX6) was identified (Figure 1 A) [7] that contains the same Ser-His-Asp catalytic triad and typical a/b hydrolase fold motif as DAGL-a. In this cocrystal structure, the nucleophilic Ser of the enzyme is covalently attached to the carbonyl moiety of the lactone. We reconstituted the ester to form the b-lactone to recover the active warhead of THL. After optimization of the geometry of the lactone, the resulting conformation was used to generate two pharmacophore models (Figure...

The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpathway. Although rad26 disruption mutants are defective in transcription-coupled repair, they are not UV sensitive. However, double mutants of RAD26 with the global genome repair determinants RAD7 and RAD16 appeared more UV sensitive than the single rad7 or rad16 mutants but not as sensitive as completely NER-deficient mutants. These findings unmask a role of RAD26 and transcription-coupled repair in UV survival, indicate that transcription-coupled repair and global genome repair are partially overlapping, and provide evidence for a residual NER modality in the double mutants. Analysis of dimer removal from the active RPB2 gene in the rad7/16 rad26 double mutants revealed (i) a contribution of the global genome repair factors Rad7p and Rad16p to repair of the transcribed strand, confirming the partial overlap between both NER subpathways, and (ii) residual repair specifically of the transcribed strand. To investigate the transcription dependence of this repair activity, strand-specific repair of the inducible GAL7 gene was investigated. The template strand of this gene was repaired only under induced conditions, pointing to a role for transcription in the residual repair in the double mutants and suggesting that transcription-coupled repair can to some extent operate independently from Rad26p. Our findings also indicate locus heterogeneity for the dependence of transcription-coupled repair on RAD26.