Assembly of KU and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at DNA double strand breaks (DS6s) forms DNA-PK holoenzyme as a critical initiating step for non-homologous end joining (NHEJ) repair of DSBs produced by radiation and chemotherapies. Advanced cryo-electron microscopy (cryo-EM) imaging together with breakthrough macromolecular X-ray crystal (MX) structures of KU and DNA-PKcs recently enabled visualization of the similar to 600 kDa DNA-PK assembly at near atomic resolution. These important static structures provide the foundation for definition and interpretation of functional movements crucial to mechanistic understanding that can be tested through solution state structure analysis. We herein therefore leverage Cryo-EM and MX structures for the interpretation of synchrotron small-angle X-ray scattering (SAXS) data on DNA-PK conformations in solution to inform the structural mechanism for NHEJ initiation. SAXS, which measures thermodynamic solution-state conformational states and assemblies outside of cryo- and solid-state conditions, unveils the inherent flexibility of KU, DNA-PKcs and DNA-PK. The combined structural measurements reveal mobility of KU80 C-terminal region (KU8OCTR), motion/plasticity of HEAT (DNA-PKcs Huntingtin, Elongation Factor 3, PP2 A, and TOR1) regions, allosteric switching upon DNA-PKcs autophosphorylation, and dimeric arrangements of DNA-PK assembly. Importantly, the results uncover displacement of the N-terminal HEAT domain during autophosphorylation as suitable for a regulated release mechanism of DNA-PKcs from DNA-PK to control unproductive access to toxic and mutagenic DNA repair intermediates. These integrated analyses show that the marriage of SAXS with cryo-EM leverages the strengths of both techniques to enable assessment of functional conformations and flexibility defining atomic-resolution molecular mechanisms for DSB repair. (C) 2020 The Authors. Published by Elsevier Ltd.

Loss of the ataxia-telangiectasia mutated (ATM) kinase causes cerebellum-specific neurodegeneration in humans. We previously demonstrated that deficiency in ATM activation via oxidative stress generates insoluble protein aggregates in human cells, reminiscent of protein dysfunction in common neurodegenerative disorders. Here, we show that this process is driven by poly-ADP-ribose polymerases (PARPs) and that the insoluble protein species arise from intrinsically disordered proteins associating with PAR-associated genomic sites in ATM-deficient cells. The lesions implicated in this process are single-strand DNA breaks dependent on reactive oxygen species, transcription, and R-loops. Human cells expressing Mre11 A-T-like disorder mutants also show PARP-dependent aggregation identical to ATM deficiency. Lastly, analysis of A-T patient cerebellum samples shows widespread protein aggregation as well as loss of proteins known to be critical in human spinocerebellar ataxias that is not observed in neocortex tissues. These results provide a hypothesis accounting for loss of protein integrity and cerebellum function in A-T.

Small cell lung cancer (SCLC) is a highly aggressive malignancy with poor outcomes associated with resistance to cisplatin-based chemotherapy. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2), which silences transcription through trimethylation of histone H3 lysine 27 (H3K27me3) and has emerged as an important therapeutic target with inhibitors targeting its methyltransferase activity under clinical investigation. Here, we show that EZH2 has a non-catalytic and PRC2-independent role in stabilizing DDB2 to promote nucleotide excision repair (NER) and govern cisplatin resistance in SCLC. Using a synthetic lethality screen, we identified important regulators of cisplatin resistance in SCLC cells, including EZH2. EZH2 depletion causes cellular cisplatin and UV hypersensitivity in an epistatic manner with DDB1-DDB2. EZH2 complexes with DDB1-DDB2 and promotes DDB2 stability by impairing its ubiquitination independent of methyltransferase activity or PRC2, thereby facilitating DDB2 localization to cyclobutane pyrimidine dimer crosslinks to govern their repair. Furthermore, targeting EZH2 for depletion with DZNep strongly sensitizes SCLC cells and tumors to cisplatin. Our findings reveal a non-catalytic and PRC2-independent function for EZH2 in promoting NER through DDB2 stabilization, suggesting a rationale for targeting EZH2 beyond its catalytic activity for overcoming cisplatin resistance in SCLC.

DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.

Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival.

A hallmark of cancer is genomic instability, which can enable cancer cells to evade therapeutic strategies. Here we employed a computational approach to uncover mechanisms underlying cancer mutational burden by focusing upon relationships between 1) translocation breakpoints and the thousands of G4 DNA-forming sequences within retrotransposons impacting transcription and exemplifying probable non-B DNA structures and 2) transcriptome profiling and cancer mutations. We determined the location and number of G4 DNA-forming sequences in the Genome Reference Consortium Human Build 38 and found a total of 358,605 covering similar to 13.4 million bases. By analyzing >97,000 unique translocation breakpoints from the Catalogue Of Somatic Mutations In Cancer (COSMIC), we found that breakpoints are overrepresented at G4 DNA-forming sequences within hominid-specific SVA retrotransposons, and generally occur in tumors with mutations in tumor suppressor genes, such as TP53. Furthermore, correlation analyses between mRNA levels and exome mutational loads from The Cancer Genome Atlas (TCGA) encompassing >450,000 gene-mutation regressions revealed strong positive and negative associations, which depended upon tissue of origin. The strongest positive correlations originated from genes not listed as cancer genes in COSMIC; yet, these show strong predictive power for survival in most tumor types by Kaplan-Meier estimation. Thus, correlation analyses of DNA structure and gene expression with mutation loads complement and extend more traditional approaches to elucidate processes shaping genomic instability in cancer. The combined results point to G4 DNA, activation of cell cycle/DNA repair pathways, and mitochondrial dysfunction as three major factors driving the accumulation of somatic mutations in cancer cells. (C) 2019 The Authors. Published by Elsevier Ltd.

Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.

Besides critical and well-studied roles in maintaining genome stability, DNA repair pathways including base excision repair (BER) are also employed by mammalian cells for targeted events such as the generation of diverse antibody repertoires and the differentiation of pluripotent cells. In this issue of The EMBO Journal, Steinacher et al () provide novel mechanistic insights into the complex role of SUMOylation of key BER proteins during active DNA demethylation-a role they demonstrate to be important for cellular differentiation and that has broader implications for BER as a whole.