DNA damage-induced dynamic changes in abundance and cytosol-nuclear translocation of proteins involved in translational processes, metabolism, and autophagy

Ionizing radiation (IR) causes DNA double-strand breaks (DSBs) and activates a versatile cellular response regulating DNA repair, cell-cycle progression, transcription, DNA replication and other processes. In recent years proteomics has emerged as a powerful tool deepening our understanding of this multifaceted response. In this study we use SILAC-based proteomics to specifically investigate dynamic changes in cytoplasmic protein abundance after ionizing radiation; we present in-depth bioinformatics analysis and show that levels of proteins involved in autophagy (cathepsins and other lysosomal proteins), proteasomal degradation (Ubiquitin-related proteins), energy metabolism (mitochondrial proteins) and particularly translation (ribosomal proteins and translation factors) are regulated after cellular exposure to ionizing radiation. Downregulation of no less than 68 ribosomal proteins shows rapid changes in the translation pattern after IR. Additionally, we provide evidence of compartmental cytosol-nuclear translocation of numerous DNA damage related proteins using protein correlation profiling. In conclusion, these results highlight unexpected cytoplasmic processes actively orchestrated after genotoxic insults and protein translocation from the cytoplasm to the nucleus as a fundamental regulatory mechanism employed to aid cell survival and preservation of genome integrity.</p

RNF168 Binds and Amplifies Ubiquitin Conjugates on Damaged Chromosomes to Allow Accumulation of Repair Proteins

SummaryDNA 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

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

The CHD4 helicase is identified as a new component of the genome surveillance machinery in a proteomic screen for factors enriched on chromatin after ionizing radiation (see also related paper by Smeenk et al. in this issue)

Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool. To further enhance the agronomic potential of rye, we produced a chromosome-scale annotated assembly of the 7.9-gigabase rye genome and extensively validated its quality by using a suite of molecular genetic resources. We demonstrate applications of this resource with a broad range of investigations. We present findings on cultivated rye's incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution, pathogen resistance, low-temperature tolerance, fertility control systems for hybrid breeding and the yield benefits of rye-wheat introgressions.Peer reviewe

Nucleolar responses to DNA double-strand breaks

Maintenance of cellular homeostasis is key to prevent transformation and disease. The cellular response to DNA double-strand breaks, primarily orchestrated by the ATM/ATR kinases is one of many mechanisms that serve to uphold genome stability and homeostasis. Upon detection of double-strand breaks (DSBs), several signaling cascades are activated to halt cell cycle progression and initiate repair. Furthermore, the DNA damage response (DDR) controls cellular processes such as transcription, splicing and metabolism. Recent studies have uncovered aspects of how the DDR operates within nucleoli. It appears that the DDR controls transcription in the nucleoli, not only when DNA breaks occur in the rDNA repeats, but also when a nuclear DDR is activated. In addition, we have gained first insights into how repair of DSBs is organized in the nucleolus. Collectively, these recent studies provide a more comprehensive picture of how the DDR regulates basic cellular functions to maintain cellular homeostasis. In this review we will summarize recent findings and discuss their implications for our understanding of how the DDR regulates transcription and repair in the nucleolus

ATR Prohibits Replication Catastrophe by Preventing Global Exhaustion of RPA

SummaryATR, 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