Insight in the multilevel regulation of NER.

Nucleotide excision repair (NER) is a key component of the DNA damage response (DDR) and it is essential to safeguard genome integrity against genotoxic insults. The regulation of NER is primarily mediated by protein post-translational modifications (PTMs). The NER machinery removes a wide spectrum of DNA helix distorting lesions, including those induced by solar radiation, through two sub-pathways: global genome nucleotide excision repair (GG-NER) and transcription coupled nucleotide excision repair (TC-NER). Severe clinical consequences associated with inherited NER defects, including premature ageing, neurodegeneration and extreme cancer-susceptibility, underscore the biological relevance of NER. In the last two decades most of the core NER machinery has been elaborately described, shifting attention to molecular mechanisms that either facilitate NER in the context of chromatin or promote the timely and accurate interplay between NER factors and various post-translational modifications. In this review, we summarize and discuss the latest findings in NER. In particular, we focus on emerging factors and novel molecular mechanisms by which NER is regulated.

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species.

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

Extreme calorie restriction and energy source starvation in Saccharomyces cerevisiae represent distinct physiological states.

Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from reserve carbohydrates were 2-3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae.

A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair.

Bulky DNA lesions in transcribed strands block RNA polymerase II (RNAPII) elongation and induce a genome-wide transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but how transcription is restored in the genome following DNA repair remains unresolved. Here, we find that the TCR-specific CSB protein loads the PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. Although dispensable for TCR-mediated repair, PAF1C is essential for transcription recovery after UV irradiation. We find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

TRiC controls transcription resumption after UV damage by regulating Cockayne syndrome protein A.

Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA-DDB1-CUL4A-RBX1 cullin-RING ubiquitin ligase complex (CRLCSA). Despite its vital role in TC-NER, little is known about the regulation of the CRLCSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin. TRiC's binding to CSA ensures its stability and DDB1-dependent assembly into the CRLCSA complex. Consequently, loss of TRiC leads to mislocalization and depletion of CSA, as well as impaired transcription recovery following UV damage, suggesting defects in TC-NER. Furthermore, Cockayne syndrome (CS)-causing mutations in CSA lead to increased TRiC binding and a failure to compose the CRLCSA complex. Thus, we uncover CSA as a TRiC substrate and reveal that TRiC regulates CSA-dependent TC-NER and the development of CS.

How Dextran Sulfate Affects C1-inhibitor Activity: A Model for Polysaccharide Potentiation.

C1-inhibitor is a key inhibitor of the complement and contact activation systems, and mutations in the protein can cause hereditary angioedema. Through an unknown mechanism, polysaccharides can increase C1-inhibitor activity against some of its target proteases. Here we present the crystal structures of the serine protease inhibitor (serpin) domain of active C1-inhibitor by itself and in complex with dextran sulfate. Unlike previously described interactions of serpins with polysaccharides, the structures and isothermal titration calorimetry experiments together reveal that dextran sulfate binds to C1-inhibitor's F1 helix with low affinity and does not invoke an allosteric change. Furthermore, one dextran sulfate molecule can bind multiple C1-inhibitor molecules. We propose that in a C1-inhibitor/protease/polysaccharide ternary complex, negatively charged polysaccharides link C1-inhibitor's positively charged F1 helix to positively charged autolysis loops of proteases. The proposed mechanism elegantly explains previous experiments showing that polysaccharide potentiation is increased against proteases with a greater positive charge in their autolysis loop.