Adenosine diphosphate (ADP)‐ribosylation is a post‐translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ...ADP‐ribosylation reactions are the poly(ADP‐ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP‐ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP‐ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP‐ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP‐interacting protein that removes mono(ADP‐ribosyl)ation on glutamate amino acid residues in PARP‐modified proteins. X‐ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl‐(ADP‐ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP‐ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.
Crystal structure and biochemical data reveal a gene mutated in patients with severe neurodegeneration to encode an elusive enzyme for removing ADP‐ribose from proteins.
Topoisomerase 2 (TOP2) DNA transactions proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer ...TOP2 drugs. How genotoxic TOP2 DNA-protein cross-links are resolved is unclear. We found that the SUMO (small ubiquitin-related modifier) ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a “split-SIM” SUMO2 engagement platform. These findings uncover a ZATT-TDP2–catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.
DNA strand breaks present a complex challenge for our cells, and the integrity of the DNA damage response machinery is critical for preventing cancer, premature aging, and neurodegenerative syndromes ...amongst other ailments. This multi-author review issue presents emerging topics relevant to understanding the fundamental structural mechanisms of DNA strand break sensing, signaling, and repair.
DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA ...substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1
variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1
Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1
normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1
high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.
The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA ...topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA–protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic “poisons” perturbs the TOP2 reaction cycle, leading to an accumulation of DNA–protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATT
Znf451
-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.
The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through ...undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT
1-BRCT
2 domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.
Vertebrate CtIP, and its fission yeast (Ctp1), budding yeast (Sae2) and plant (Com1) orthologs have emerged as key regulatory molecules in cellular responses to DNA double strand breaks (DSBs). By ...modulating the nucleolytic 5′-3′ resection activity of the Mre11/Rad50/Nbs1 (MRN) DSB repair processing and signaling complex, CtIP/Ctp1/Sae2/Com1 is integral to the channeling of DNA double strand breaks through DSB repair by homologous recombination (HR). Nearly two decades since its discovery, emerging new data are defining the molecular underpinnings for CtIP DSB repair regulatory activities. CtIP homologs are largely intrinsically unstructured proteins comprised of expanded regions of low complexity sequence, rather than defined folded domains typical of DNA damage metabolizing enzymes and nucleases. A compact structurally conserved N-terminus forms a functionally critical tetrameric helical dimer of dimers (THDD) region that bridges CtIP oligomers, and is flexibly appended to a conserved C-terminal Sae2-homology DNA binding and DSB repair pathway choice regulatory hub which influences nucleolytic activities of the MRN core nuclease complex. The emerging evidence from structural, biophysical, and biological studies converges on CtIP having functional roles in DSB repair that include: 1) dynamic DNA strand coordination through direct DNA binding and DNA bridging activities, 2) MRN nuclease complex cofactor functions that direct MRN endonucleolytic cleavage of protein-blocked DSB ends and 3) acting as a protein binding hub targeted by the cell cycle regulatory apparatus, which influences CtIP expression and activity via layers of post-translational modifications, protein–protein interactions and DNA binding.
ABSTRACT
Alveolar epithelial cell (AEC) mitochondrial dysfunction and apoptosis are important in idiopathic pulmonary fibrosis and asbestosis. Sirtuin 3 (SIRT3) detoxifies mitochondrial reactive ...oxygen species, in part, by deacetylating manganese superoxide dismutase (MnSOD) and mitochondrial 8‐oxoguanine DNA glycosylase. We reasoned that SIRT3 deficiency occurs in fibrotic lungs and thereby augments AEC mtDNA damage and apoptosis. Human lungs were assessed by using immunohistochemistry for SIRT3 activity via acetylated MnSODK68. Murine AEC SIRT3 and cleaved caspase‐9 (CC‐9) expression were assayed by immunoblotting with or without SIRT3 enforced expression or silencing. mtDNA damage was measured by using quantitative PCR and apoptosis via ELISA. Pulmonary fibrosis after asbestos or bleomycin exposure was evaluated in 129SJ/wild‐type and SIRT3‐knockout mice (Sirt3−/−) by using fibrosis scoring and lung collagen levels. Idiopathic pulmonary fibrosis lung alveolar type II cells have increased MnSODK68 acetylation compared with controls. Asbestos and H2O2 diminished AEC SIRT3 protein expression and increased mitochondrial protein acetylation, including MnSODK68. SIRT3 enforced expression reduced oxidant‐induced AEC OGG1K338/341 acetylation, mtDNA damage, and apoptosis, whereas SIRT3 silencing promoted these effects. Asbestos‐ or bleomycin‐induced lung fibrosis, AEC mtDNA damage, and apoptosis in wild‐type mice were amplified in Sirt3−/− animals. These data suggest a novel role for SIRT3 deficiency in mediating AEC mtDNA damage, apoptosis, and lung fibrosis.—Jablonski, R. P., Kim, S.‐J., Cheresh, P., Williams, D. B., Morales‐Nebreda, L., Cheng, Y., Yeldandi, A., Bhorade, S., Pardo, A., Selman, M., Ridge, K., Gius, D., Budinger, G. R. S., Kamp, D. W. SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis. FASEB J. 31, 2520–2532 (2017). www.fasebj.org
The CDC biofilm reactor is a robust culture system with high reproducibility in which biofilms can be grown for a wide variety of analyses. Multiple material types are available as growth substrates, ...yet data from biofilms grown on biologically relevant materials is scarce, particularly for antibiotic efficacy against differentially supported biofilms. In this study, CDC reactor holders were modified to allow growth of biofilms on collagen, a biologically relevant substrate. Susceptibility to multiple antibiotics was compared between biofilms of varying species grown on collagen versus standard polycarbonate coupons. Data indicated that in 13/18 instances, biofilms on polycarbonate were more susceptible to antibiotics than those on collagen, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. These outcomes may influence the translatability of antibiotic susceptibility profiles that have been collected for biofilms on hard plastic materials. Data may also help to advance information on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Mre11‐Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP‐driven states regulate the sensing, processing and signaling of DNA double‐strand breaks are largely unknown. Here ...we design structure‐based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP‐driven movements within the catalytic domains. With this strategy we identify Rad50 separation‐of‐function mutants that either promote or destabilize the ATP‐bound state. Crystal structures, X‐ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP‐induced ‘closed’ conformation promotes DNA end binding and end tethering, while hydrolysis‐induced opening is essential for DNA resection. Reducing the stability of the ATP‐bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double‐strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11‐Rad50‐Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure‐based mutational analyses. These collective results suggest that ATP‐dependent Rad50 conformations switch the Mre11‐Rad50 complex between DNA tethering, ATM signaling, and 5′ strand resection, revealing molecular mechanisms regulating responses to DNA double‐strand breaks.
Synopsis
Comprehensive structural and functional studies with Rad50 separation‐of‐function mutants show how ATP binding and hydrolysis toggle the Mre11‐Rad50‐Nbs1 (MRN) complex between closed conformations promoting DNA end tethering, and open states mediating end resection.
Structure‐based mutations in Rad50 alter protein core plasticity and stability of the ATP‐bound state.
The ATP‐bound ‘closed’ state of MR promotes DNA binding and end tethering.
ATP hydrolysis‐induced opening of the MR complex is necessary for resection nuclease activity.
A balance between the ‘closed’ and ‘open’ states is essential for DNA repair and ATM checkpoint activation.
Comprehensive structural and functional studies with Rad50 separation‐of‐function mutants show how ATP binding and hydrolysis toggle the MRN complex between closed conformations promoting DNA end tethering, and open states mediating end resection.
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