Structural basis for RNA polymerase II ubiquitylation and inactivation in transcription-coupled repair.

During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryogenic electron microscopy, biochemical assays and cell biology approaches, we found that ELOF1 serves as an adaptor to stably position UVSSA and CRL4CSA on arrested Pol II, leading to ligase neddylation and activation of Pol II ubiquitylation. In the presence of ELOF1, a transcription factor IIS (TFIIS)-like element in UVSSA gets ordered and extends through the Pol II pore, thus preventing reactivation of Pol II by TFIIS. Our results provide the structural basis for Pol II ubiquitylation and inactivation in TCR.

Avidity-based biosensors for ubiquitylated PCNA reveal choreography of DNA damage bypass.

In eukaryotes, the posttranslational modifier ubiquitin is used to regulate the amounts, interactions, or activities of proteins in diverse pathways and signaling networks. Its effects are mediated by monoubiquitin or polyubiquitin chains of varying geometries. We describe the design, validation, and application of a series of avidity-based probes against the ubiquitylated forms of the DNA replication clamp, proliferating cell nuclear antigen (PCNA), in budding yeast. Directed against total ubiquitylated PCNA or specifically K63-polyubiquitylated PCNA, the probes are tunable in their activities and can be used either as biosensors or as inhibitors of the PCNA-dependent DNA damage bypass pathway. Used in live cells, the probes revealed the timing of PCNA ubiquitylation during damage bypass and a particular susceptibility of the ribosomal DNA locus to the activation of the pathway. Our approach is applicable to a wide range of ubiquitin-conjugated proteins, thus representing a generalizable strategy for the design of biosensors for specific (poly)ubiquitylated forms of individual substrates.

Transcription and replication: walking a genomic tightrope hand-in-hand.

A recent study by Fenstermaker et al. in Nature describes how transcriptionally active RNA polymerase II (Pol II) clings to the genomic tightrope during the passage of the replication fork and rapidly resumes transcription of immature RNA from both strands of nascent DNA, facilitated by protein-protein interactions between the replication and transcription machineries.

Linkage reprogramming by tailor-made E3s reveals polyubiquitin chain requirements in DNA-damage bypass.

A polyubiquitin chain can adopt a variety of shapes, depending on how the ubiquitin monomers are joined. However, the relevance of linkage for the signaling functions of polyubiquitin chains is often poorly understood because of our inability to control or manipulate this parameter in vivo. Here, we present a strategy for reprogramming polyubiquitin chain linkage by means of tailor-made, linkage- and substrate-selective ubiquitin ligases. Using the polyubiquitylation of the budding yeast replication factor PCNA in response to DNA damage as a model case, we show that altering the features of a polyubiquitin chain in vivo can change the fate of the modified substrate. We also provide evidence for redundancy between distinct but structurally similar linkages, and we demonstrate by proof-of-principle experiments that the method can be generalized to targets beyond PCNA. Our study illustrates a promising approach toward the in vivo analysis of polyubiquitin signaling.

NBS1 interacts with Notch signaling in neuronal homeostasis.

NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.

RNase H1 and H2 Are Differentially Regulated to Process RNA-DNA Hybrids.

RNA-DNA hybrids are tightly regulated to ensure genome integrity. The RNase H enzymes RNase H1 and H2 contribute to chromosomal stability through the removal of RNA-DNA hybrids. Loss of RNase H2 function is implicated in human diseases of the nervous system and cancer. To better understand RNA-DNA hybrid dynamics, we focused on elucidating the regulation of the RNase H enzymes themselves. Using yeast as a model system, we demonstrate that RNase H1 and H2 are controlled in different manners. RNase H2 has strict cell cycle requirements, in that it has an essential function in G2/M for both R-loop processing and ribonucleotide excision repair. RNase H1, however, can function independently of the cell cycle to remove R-loops and appears to become activated in response to high R-loop loads. These results provide us with a more complete understanding of how and when RNA-DNA hybrids are acted upon by the RNase H enzymes.