Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity

• Published in: Molecular cell 2017-09, Vol.67 (5), p.882-890.e5
• Main Authors: Vujanovic, Marko, Krietsch, Jana, Raso, Maria Chiara, Terraneo, Nastassja, Zellweger, Ralph, Schmid, Jonas A., Taglialatela, Angelo, Huang, Jen-Wei, Holland, Cory L., Zwicky, Katharina, Herrador, Raquel, Jacobs, Heinz, Cortez, David, Ciccia, Alberto, Penengo, Lorenza, Lopes, Massimo
• Format: Article
• Language: English
• Subjects: Animals; cancer chemotherapeutics; CRISPR-Cas Systems; DNA Damage; DNA damage tolerance; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Replication; DNA, Neoplasm - biosynthesis; DNA, Neoplasm - genetics; DNA, Neoplasm - ultrastructure; electron microscopy in vivo; HCT116 Cells; HEK293 Cells; Humans; Kinetics; Mice; Mutation; Neoplasms - enzymology; Neoplasms - genetics; Neoplasms - ultrastructure; PCNA ubiquitination; Polyubiquitin - metabolism; postreplication repair; Proliferating Cell Nuclear Antigen - genetics; Proliferating Cell Nuclear Antigen - metabolism; replication fork progression; replication fork reversal; Replication Origin; RNA Interference; Short; single-molecule approaches; Transfection; Ubiquitin-Conjugating Enzymes - genetics; Ubiquitin-Conjugating Enzymes - metabolism; Ubiquitination; ZRANB3 DNA translocase
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2017.08.010

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy. [Display omitted] •Fork slowing and reversal upon damage require K63-linked PCNA polyubiquitination•ZRANB3 mediates fork slowing/reversal in vivo via binding to polyubiquitinated PCNA•ZRANB3 DNA translocase—not nuclease—activity mediates fork slowing and reversal•Mammalian error-free postreplication repair entails global fork slowing and reversal Vujanovic et al. show that UBC13-mediated, K63-linked PCNA polyubiquitination mediates DNA damage-induced replication fork slowing and reversal, via recruitment to forks of ZRANB3 DNA translocase. These data link the postreplication repair pathway, yet elusive in mammals, to the regulation of fork progression and remodeling.