Discovery and Characterization of ZUFSP/ZUP1, a Distinct Deubiquitinase Class Important for Genome Stability.

Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.

Regulation of PCNA-protein interactions for genome stability.

Proliferating cell nuclear antigen (PCNA) has a central role in promoting faithful DNA replication, providing a molecular platform that facilitates the myriad protein-protein and protein-DNA interactions that occur at the replication fork. Numerous PCNA-associated proteins compete for binding to a common surface on PCNA; hence these interactions need to be tightly regulated and coordinated to ensure proper chromosome replication and integrity. Control of PCNA-protein interactions is multilayered and involves post-translational modifications, in particular ubiquitylation, accessory factors and regulated degradation of PCNA-associated proteins. This regulatory framework allows cells to maintain a fine-tuned balance between replication fidelity and processivity in response to DNA damage.

Serine ADP-Ribosylation Depends on HPF1.

ADP-ribosylation (ADPr) regulates important patho-physiological processes through its attachment to different amino acids in proteins. Recently, by precision mapping on all possible amino acid residues, we identified histone serine ADPr marks in the DNA damage response. However, the biochemical basis underlying this serine modification remained unknown. Here we report that serine ADPr is strictly dependent on histone PARylation factor 1 (HPF1), a recently identified regulator of PARP-1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. Moreover, adding HPF1 to in vitro PARP-1/PARP-2 reactions is necessary and sufficient for serine-specific ADPr of histones and PARP-1 itself. Three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on HMG proteins and hundreds of other targets indicates that serine ADPr is a widespread modification. We propose that O-linked protein ADPr is the key signal in PARP-1/PARP-2-dependent processes that govern genome stability.

HPF1/C4orf27 Is a PARP-1-Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity.

We report the identification of histone PARylation factor 1 (HPF1; also known as C4orf27) as a regulator of ADP-ribosylation signaling in the DNA damage response. HPF1/C4orf27 forms a robust protein complex with PARP-1 in cells and is recruited to DNA lesions in a PARP-1-dependent manner, but independently of PARP-1 catalytic ADP-ribosylation activity. Functionally, HPF1 promotes PARP-1-dependent in trans ADP-ribosylation of histones and limits DNA damage-induced hyper-automodification of PARP-1. Human cells lacking HPF1 exhibit sensitivity to DNA damaging agents and PARP inhibition, thereby suggesting an important role for HPF1 in genome maintenance and regulating the efficacy of PARP inhibitors. Collectively, our results demonstrate how a fundamental step in PARP-1-dependent ADP-ribosylation signaling is regulated and suggest that HPF1 functions at the crossroads of histone ADP-ribosylation and PARP-1 automodification.

SLX4: not SIMply a nuclease scaffold?

In this issue, Guervilly et al. (2015) and Ouyang et al. (2015) identify SUMO-interacting motifs (SIMs) in the SLX4 DNA repair nuclease scaffold protein that promote its functions in genome stability maintenance pathways independently of its ubiquitin-binding properties.

Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage.

We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage.

C16orf72/HAPSTR1/TAPR1 functions with BRCA1/Senataxin to modulate replication-associated R-loops and confer resistance to PARP disruption.

While the toxicity of PARP inhibitors to cells with defects in homologous recombination (HR) is well established, other synthetic lethal interactions with PARP1/PARP2 disruption are poorly defined. To inform on these mechanisms we conducted a genome-wide screen for genes that are synthetic lethal with PARP1/2 gene disruption and identified C16orf72/HAPSTR1/TAPR1 as a novel modulator of replication-associated R-loops. C16orf72 is critical to facilitate replication fork restart, suppress DNA damage and maintain genome stability in response to replication stress. Importantly, C16orf72 and PARP1/2 function in parallel pathways to suppress DNA:RNA hybrids that accumulate at stalled replication forks. Mechanistically, this is achieved through an interaction of C16orf72 with BRCA1 and the RNA/DNA helicase Senataxin to facilitate their recruitment to RNA:DNA hybrids and confer resistance to PARP inhibitors. Together, this identifies a C16orf72/Senataxin/BRCA1-dependent pathway to suppress replication-associated R-loop accumulation, maintain genome stability and confer resistance to PARP inhibitors.

Tools for Decoding Ubiquitin Signaling in DNA Repair.

The maintenance of genome stability requires dedicated DNA repair processes and pathways that are essential for the faithful duplication and propagation of chromosomes. These DNA repair mechanisms counteract the potentially deleterious impact of the frequent genotoxic challenges faced by cells from both exogenous and endogenous agents. Intrinsic to these mechanisms, cells have an arsenal of protein factors that can be utilised to promote repair processes in response to DNA lesions. Orchestration of the protein factors within the various cellular DNA repair pathways is performed, in part, by post-translational modifications, such as phosphorylation, ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs). In this review, we firstly explore recent advances in the tools for identifying factors involved in both DNA repair and ubiquitin signaling pathways. We then expand on this by evaluating the growing repertoire of proteomic, biochemical and structural techniques available to further understand the mechanistic basis by which these complex modifications regulate DNA repair. Together, we provide a snapshot of the range of methods now available to investigate and decode how ubiquitin signaling can promote DNA repair and maintain genome stability in mammalian cells.

Serine-linked PARP1 auto-modification controls PARP inhibitor response.

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are recruited and activated by DNA damage, resulting in ADP-ribosylation at numerous sites, both within PARP1 itself and in other proteins. Several PARP1 and PARP2 inhibitors are currently employed in the clinic or undergoing trials for treatment of various cancers. These drugs act primarily by trapping PARP1 on damaged chromatin, which can lead to cell death, especially in cells with DNA repair defects. Although PARP1 trapping is thought to be caused primarily by the catalytic inhibition of PARP-dependent modification, implying that ADP-ribosylation (ADPr) can counteract trapping, it is not known which exact sites are important for this process. Following recent findings that PARP1- or PARP2-mediated modification is predominantly serine-linked, we demonstrate here that serine ADPr plays a vital role in cellular responses to PARP1/PARP2 inhibitors. Specifically, we identify three serine residues within PARP1 (499, 507, and 519) as key sites whose efficient HPF1-dependent modification counters PARP1 trapping and contributes to inhibitor tolerance. Our data implicate genes that encode serine-specific ADPr regulators, HPF1 and ARH3, as potential PARP1/PARP2 inhibitor therapy biomarkers.

Discovery of a Selective Allosteric Inhibitor Targeting Macrodomain 2 of Polyadenosine-Diphosphate-Ribose Polymerase 14.

Macrodomains are conserved protein interaction modules that can be found in all domains of life including in certain viruses. Macrodomains mediate recognition of sequence motifs harboring adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation studies so far. Here, we describe an efficient screening strategy for identification of small molecule inhibitors that displace ADPR from macrodomains. We report the discovery and characterization of a macrodomain inhibitor, GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low micromolar affinity. Co-crystallization of a GeA-69 analogue with PARP14 MD2 revealed an allosteric binding mechanism explaining its selectivity over other human macrodomains. We show that GeA-69 engages PARP14 MD2 in intact cells and prevents its localization to sites of DNA damage.

MRNIP/C5orf45 Interacts with the MRN Complex and Contributes to the DNA Damage Response.

Through an RNAi-based screen for previously uncharacterized regulators of genome stability, we have identified the human protein C5orf45 as an important factor in preventing the accumulation of DNA damage in human cells. Here, we functionally characterize C5orf45 as a binding partner of the MRE11-RAD50-NBS1 (MRN) damage-sensing complex. Hence, we rename C5orf45 as MRNIP for MRN-interacting protein (MRNIP). We find that MRNIP is rapidly recruited to sites of DNA damage. Cells depleted of MRNIP display impaired chromatin loading of the MRN complex, resulting in reduced DNA end resection and defective ATM-mediated DNA damage signaling, a reduced ability to repair DNA breaks, and radiation sensitivity. Finally, we show that MRNIP phosphorylation on serine 115 leads to its nuclear localization, and this modification is required for MRNIP's role in promoting genome stability. Collectively, these data reveal that MRNIP is an important component of the human DNA damage response.

Lamin A/C-dependent interaction with 53BP1 promotes cellular responses to DNA damage.

Lamins A/C have been implicated in DNA damage response pathways. We show that the DNA repair protein 53BP1 is a lamin A/C binding protein. In undamaged human dermal fibroblasts (HDF), 53BP1 is a nucleoskeleton protein. 53BP1 binds to lamins A/C via its Tudor domain, and this is abrogated by DNA damage. Lamins A/C regulate 53BP1 levels and consequently lamin A/C-null HDF display a 53BP1 null-like phenotype. Our data favour a model in which lamins A/C maintain a nucleoplasmic pool of 53BP1 in order to facilitate its rapid recruitment to sites of DNA damage and could explain why an absence of lamin A/C accelerates aging.

DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks.

Ubiquitin-mediated processes orchestrate critical DNA-damage signaling and repair pathways. We identify human DVC1 (C1orf124; Spartan) as a cell cycle-regulated anaphase-promoting complex (APC) substrate that accumulates at stalled replication forks. DVC1 recruitment to sites of replication stress requires its ubiquitin-binding UBZ domain and PCNA-binding PIP box motif but is independent of RAD18-mediated PCNA monoubiquitylation. Via a conserved SHP box, DVC1 recruits the ubiquitin-selective chaperone p97 to blocked replication forks, which may facilitate p97-dependent removal of translesion synthesis (TLS) DNA polymerase η (Pol η) from monoubiquitylated PCNA. DVC1 knockdown enhances UV light-induced mutagenesis, and depletion of human DVC1 or the Caenorhabditis elegans ortholog DVC-1 causes hypersensitivity to replication stress-inducing agents. Our findings establish DVC1 as a DNA damage-targeting p97 adaptor that protects cells from deleterious consequences of replication blocks and suggest an important role of p97 in ubiquitin-dependent regulation of TLS.

Conserved cysteine residues in the mammalian lamin A tail are essential for cellular responses to ROS generation.

Pre-lamin A and progerin have been implicated in normal aging, and the pathogenesis of age-related degenerative diseases is termed 'laminopathies'. Here, we show that mature lamin A has an essential role in cellular fitness and that oxidative damage to lamin A is involved in cellular senescence. Primary human dermal fibroblasts (HDFs) aged replicatively or by pro-oxidants acquire a range of dysmorphic nuclear shapes. We observed that conserved cysteine residues in the lamin A tail domain become hyperoxidized in senescent fibroblasts, which inhibits the formation of lamin A inter- and intramolecular disulfide bonds. Both in the absence of lamin A and in the presence of a lamin A cysteine-to-alanine mutant, which eliminates these cysteine residues (522, 588, and 591), mild oxidative stress induced nuclear disorganization and led to premature senescence as a result of decreased tolerance to ROS stimulators. Human dermal fibroblasts lacking lamin A or expressing the lamin A cysteine-to-alanine mutant displayed a gene expression profile of ROS-responsive genes characteristic of chronic ROS stimulation. Our findings suggest that the conserved C-terminal cysteine residues are essential for lamin A function and that loss or oxidative damage to these cysteine residues promotes cellular senescence.