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.

Nuclear myosin VI maintains replication fork stability.

The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase Werner helicase interacting protein 1 (WRNIP1) in protecting these structures from DNA2-mediated nucleolytic attack. Using functionalized affinity probes to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.

DNA-Polymer Conjugates by Photoinduced RAFT Polymerization.

Conventional grafting-to approaches to DNA-polymer conjugates are often limited by low reaction yields due to the sterically hindered coupling of a presynthesized polymer to DNA. The grafting-from strategy, in contrast, allows one to directly graft polymers from an initiator that is covalently attached to DNA. Herein, we report blue-light-mediated reversible addition-fragmentation chain-transfer (Photo-RAFT) polymerization from two different RAFT agent-terminated DNA sequences using Eosin Y as the photocatalyst in combination with ascorbic acid. Three monomer families (methacrylates, acrylates and acrylamides) were successfully polymerized from DNA employing Photo-RAFT polymerization. We demonstrate that the length of the grown polymer chain can be varied by altering the monomer to DNA-initiator ratio, while the self-assembly features of the DNA strands were maintained. In summary, we describe a convenient, light-mediated approach toward DNA-polymer conjugates via the grafting-from approach.