Nucleotide excision repair: interplay between nuclear compartmentalization, histone modifications and signaling

Maintaining the integrity of genetic information is one of the crucial functions of the cell. Depending on the type of DNA damage, there are several repair pathways dedicated to a quick and efficient repair of the damage. A major source of DNA damage is exposure to UV light, which causes formation of 6’-4’photoproducts and pyrimidine dimers. These are bulky DNA adducts that cause distortion of the helix structure. Such lesions are repaired by Nucleotide Excision Repair (NER). Nucleotide Excision Repair occurs by two sub-pathways, depending on the genomic location of the lesion. Transcription coupled NER (TC-NER) repairs lesions in actively transcribed genes, which global genomic NER (GG-NER) can repair all types of lesions. These two pathways differ in their recognition step. Lesion recognition is followed by verification of damage, excision of the damaged strand, and refilling of the gap by DNA synthesis. An important unanswered question in the field of NER is how the removal of lesions occurs in the context of chromatin structure. Recognition of lesions in heterochromatin requires a decondensation of chromatin to enable access by repair factors. Additionally, lesion recognition requires cascades of recruitment of the various proteins, in a tightly regulated and synchronized manner. We show that the recognition step during GG-NER consists of a ZRF1-DICER-MMSET axis linking lesion recognition via DDB2 to lesion verification via XPA followed by subsequent repair. ZRF1 recognizes the H2AK119 ub mark set by the UV-RING1B complex. This results in translocation of the lesion to the nucleolus, and remodeling of the complex to the UV-CUL4A complex. Formation of this complex enables the next phase of ubiquitylation that regulates NER repair proteins. ZRF1 in turn also contributed to chromatin decondensation through recruitment of DICER. DICER enables relaxation of chromatin structure in a PARP1 dependent manner. It also recruits MMSET, which sets the H4K20me2 mark. This histone mark serves as a tethering platform for recruitment of XPA, a core NER component which is essential for further repair to take place. Thus, we have discovered a novel and essential function for these proteins in NER.Eine wesentliche Aufgabe der Zelle ist es die Integrität ihrer DNA zu schützen, um die darin enthaltene genetische Information zu erhalten. Abhängig von der Art der DNA-Schädigung werden in der Zelle unterschiedliche DNA-Reparaturmechanismen angeschaltet, die eine schnelle und effiziente Reparatur des Schadens gewährleisten. Eine der zahlreichen zellulären DNA Reparaturmechanismen ist die Nukleotidexzisionsreparatur (NER), welche unterschiedliche Schäden wie zum Beispiel Cyclobutanpyrimidin Dimere (CPD) und 6-4 Photoprodukte repariert, die nach der Bestrahlung mit UV Licht entstehen. In Säugerzellen werden in der NER Schäden durch zwei unterschiedliche Mechanismen repariert. Schäden in transkribierten genomischen Bereichen werden durch die sogenannte transcription-coupled NER (TC-NER) repariert, alle anderen genomische Bereiche werden durch global genomic NER (GG-NER) repariert. Diese beiden DNA Reparaturmechanismen unterscheiden sich lediglich in der Schadenserkennung. In deren Anschluss nutzen TC-NER und GG-NER einen gemeinsamen Mechanismus zur Verifizierung, zur Exzsion des Schadens und zur Auffüllung der entstandenen etwa 30 Nukleotide umfassenden Lücke durch DNA Synthese. Wie NER im Kontext der Chromatinstruktur verläuft ist eine bislang noch offene Frage. Die Schadenserkennung im Heterochromatin benötigt eine Dekondensierung bzw. Öffnung der Chromatinkonformation, um die Rekrutierung von DNA Reparaturfaktoren zu ermöglichen. Ausserdem werden während der Schadenserkennung unterschiedliche Proteinkomplexe in einer synchronisierten bzw. zeitlich festgelegten Folge zur DNA-Läsion rekrutiert. In der vorliegenden Arbeit wird gezeigt, dass während der GG-NER die Schadenserkennung durch das Zusammenspiel der Faktoren ZRF1, DICER und MMSET mit dem DNA Reparturerkennungsfaktor DDB2 geprägt ist, welches die Rekrutierung des DNA Reparaturfaktors XPA ermöglicht. Dabei bindet ZRF1 mono-ubiquitiniertes histone H2A (H2A-K119-Ub), welches durch den UV-RING1B Komplex katalysiert wird. Derart modifiziertes Chromatin wird zum Nukleolus transloziert, wo durch molekulares Remodeling der UV-RING1B Komplex in den UV-CUL4A Komplex umgebaut wird. Die Herstellung des UV-CUL4A Komplexes leitet dann die nächste Phase von Ubiquitinierungsreaktionen ein, welche wiederum nachfolgende Reparaturreaktionen regulieren. Weiterhin ist ZRF1 an der Chromatin-Dekondensierung beteiligt, da es über die Rekruiterung von DICER eine PARP1-abhängige Relaxation der Chromatin-Konformation hervorruft. DICER wiederum ist für die Rekruiterung des Methyltransferase MMSET essentiell, welche ein Methylierung am Histone H4 (H4K20me2) bedingt. Diese Histonmodifizierung dient als eine Art Bindeplattform für den Reparaturfaktor XPA, eine der Hauptkomponenten der NER. Zusammengenommen konnte eine neue und essentielle Funktion der obengenannten Proteine bzw. deren Zusammenspiel während der NER entdeckt werden

ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair.

Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV-RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV-DDB-CUL4A E3 ligase complex (DDB1-DDB2-CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites

A semiconductor 96-microplate platform for electrical-imaging based high-throughput phenotypic screening

Abstract High-content imaging for compound and genetic profiling is popular for drug discovery but limited to endpoint images of fixed cells. Conversely, electronic-based devices offer label-free, live cell functional information but suffer from limited spatial resolution or throughput. Here, we introduce a semiconductor 96-microplate platform for high-resolution, real-time impedance imaging. Each well features 4096 electrodes at 25 µm spatial resolution and a miniaturized data interface allows 8× parallel plate operation (768 total wells) for increased throughput. Electric field impedance measurements capture >20 parameter images including cell barrier, attachment, flatness, and motility every 15 min during experiments. We apply this technology to characterize 16 cell types, from primary epithelial to suspension cells, and quantify heterogeneity in mixed co-cultures. Screening 904 compounds across 13 semiconductor microplates reveals 25 distinct responses, demonstrating the platform’s potential for mechanism of action profiling. The scalability and translatability of this semiconductor platform expands high-throughput mechanism of action profiling and phenotypic drug discovery applications

ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites