DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors.

Our paper identified nuclear proteins likely harboring disordered low-complexity sequences via precipitation by b-isox microcrystals. In Table S2, we ranked 580 nuclear proteins isolated in this manner and indicated that they were ordered according to the density of spectral counts. It has come to our attention that the proteins in this table are ordered by the relative density of [G/S]Y[G/S] triplet repeats rather than by spectral counts. This error affects the following sentence in the text of the Results section: ''Among the 580 mammalian proteins selectively precipitated by b-isox microcrystals, TAF15 registered the second highest number of spectral counts, and the largest subunit of RNA polymerase II registered the third highest number of spectral counts (Table S2).'' This is because the named positions had been based on ranking by triplet repeat density. We now provide with the article online the correctly ordered Table S2 (by spectral counts instead of triplet repeat density), and the affected sentence has now been changed to indicate the ranking positions of these proteins when ordered by spectral counts, such that TAF15 is 23 rd on the list and the largest subunit of RNA polymerase is 46 th. All proteins on the list are well above the false discovery rate, and the fact that both TAF15 and the largest subunit of RNA polymerase II are close to the very top of the list means that these adjustments do not alter any results or conclusions presented in the paper. We note that Table S3, which presents the yeast nuclear proteins precipitated by b-isox microcrystals, was correctly ordered by density of spectral counts as indicated. We wish to thank David Trudgian, a computational scientist in our Mass Sepctrometry Shared Resource Core, for pointing out the inconsistency in the organization and annotation of the original Table S2.

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.