Ronald Pak Cheung Wong

Double-strand breaks (DSBs) are critical lesions in genomic DNA, and their accurate repair is essential for maintaining genome stability. The nuclear actin cytoskeleton has been implicated in homology-directed repair (HDR) of DSBs. However, the underlying mechanism remains poorly understood. Here, we report that Myosin VI (Myo6), an actin-based motor protein, cooperates with F-actin in end resection and DSB mobilization. Our findings reveal that Myo6 directly interacts with both KU70 and the ubiquitin-dependent segregase VCP to facilitate the extraction of the KU70/80 complex from chromatin. This process is supported by F-actin, revealing an interplay between nuclear actin dynamics and the DSB repair machinery. By elucidating the function of Myo6 and its direct interactions with key repair factors, our study provides mechanistic insight into how repair mechanisms rely on nuclear actin to safeguard genome integrity.

Chromatin remodellers play a crucial role in transcription, replication and genome maintenance by dynamically regulating the accessibility of DNA. The INO80 complex contributes to multiple aspects of DNA repair and DNA replication stress management. It primarily facilitates nucleosome sliding and has been linked to the removal of the histone variant H2A.Z from chromatin. Our study demonstrates that the INO80 complex promotes DNA damage bypass through postreplicative daughter-strand gap repair. The complex acts downstream of PCNA ubiquitylation, enabling both Exo1-dependent gap expansion and gap filling via translesion synthesis or template switching. Importantly, its function in DNA damage bypass is independent of H2A.Z exchange and separable from its roles in DNA double-strand break repair and transcriptional regulation. Instead, it is consistent with a ruler-like activity that ensures proper nucleosome positioning around both ends of daughter-strand gaps, providing access to exonucleases and DNA polymerases involved in gap processing. These findings underscore the importance of the INO80 complex in facilitating access to damaged DNA regions for timely and effective repair.

DNA lesions are a threat to genome stability. To cope with them during DNA replication, cells have evolved lesion bypass mechanisms: Translesion Synthesis (TLS), which allows the cell to insert a nucleotide directly opposite the lesion, with the risk of introducing a mutation, and error-free damage avoidance (DA), which uses homologous recombination to retrieve the genetic information from the sister chromatid. In this study, we investigate the timing of lesion bypass in yeast and its implications for the accuracy of the process. Our findings reveal that DNA polymerase η can bypass common, UV-induced cyclobutane pyrimidine dimers at the fork, immediately after encountering the blocking lesion. In contrast, TLS at (6–4) photoproducts and bulky G-AAF adducts, mediated by Rev1 and Pol ζ, takes place behind the fork, at post-replicative gaps that are generated downstream of the lesion after repriming. We show that in this latter situation, TLS competes with the DA pathway, thus reducing overall mutagenicity of damage bypass. Additionally, our study demonstrates that Exo1 nuclease influences the balance between TLS and DA by modulating the size of the post-replicative gaps.

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.

DNA lesions are a threat to genome stability. To cope with them during DNA replication, cells have evolved lesion bypass mechanisms: Translesion Synthesis (TLS), which allows the cell to insert a nucleotide directly opposite the lesion, with the risk of introducing a mutation, and error-free Damage Avoidance (DA), which uses homologous recombination to retrieve the genetic information from the sister chromatid. In this study, we investigate the timing of lesion bypass and its implications for the accuracy of the process. Our findings reveal that DNA polymerase η can bypass common, UV-induced TT-cyclobutane pyrimidine dimers at the fork, immediately after encountering the blocking lesion. In contrast, TLS at TT(6-4) photoproducts and bulky G-AAF adducts, mediated by Rev1 and Pol ζ, takes place behind the fork, at post-replicative gaps that are generated downstream of the lesion after repriming. We show that in this latter situation, TLS competes with the DA pathway, thus reducing overall mutagenicity of damage bypass. Additionally, our study demonstrates that Exo1 nuclease influences the balance between TLS and DA by modulating the size of the post-replicative gaps.

Replication of damaged DNA is challenging because lesions in the replication template frequently interfere with an orderly progression of the replisome. In this situation, complete duplication of the genome is ensured by the action of DNA damage bypass pathways effecting either translesion synthesis by specialized, damage-tolerant DNA polymerases or a recombination-like mechanism called template switching (TS). Here we report that budding yeast Pif1, a helicase known to be involved in the resolution of complex DNA structures as well as the maturation of Okazaki fragments during replication, contributes to DNA damage bypass. We show that Pif1 expands regions of single-stranded DNA, so-called daughter-strand gaps, left behind the replication fork as a consequence of replisome re-priming. This function requires interaction with the replication clamp, proliferating cell nuclear antigen, facilitating its recruitment to damage sites, and complements the activity of an exonuclease, Exo1, in the processing of post-replicative daughter-strand gaps in preparation for TS. Our results thus reveal a novel function of a conserved DNA helicase that is known as a key player in genome maintenance.

Polymerase‐blocking DNA lesions are thought to elicit a checkpoint response via accumulation of single‐stranded DNA at stalled replication forks. However, as an alternative to persistent fork stalling, re‐priming downstream of lesions can give rise to daughter‐strand gaps behind replication forks. We show here that the processing of such structures by an exonuclease, Exo1, is required for timely checkpoint activation, which in turn prevents further gap erosion in S phase. This Rad9‐dependent mechanism of damage signaling is distinct from the Mrc1‐dependent, fork‐associated response to replication stress induced by conditions such as nucleotide depletion or replisome‐inherent problems, but reminiscent of replication‐independent checkpoint activation by single‐stranded DNA. Our results indicate that while replisome stalling triggers a checkpoint response directly at the stalled replication fork, the response to replication stress elicited by polymerase‐blocking lesions mainly emanates from Exo1‐processed, postreplicative daughter‐strand gaps, thus offering a mechanistic explanation for the dichotomy between replisome‐ versus template‐induced checkpoint signaling.