Structural Biology of Genome Maintenance and DNA repair

基因组维护和 DNA 修复的结构生物学

• 批准号: 8734164
• 负责人: Robert Williams
• 金额: $ 164.27万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8734164
• 关键词: ADP ribosylation; APTX gene; Adenosine Diphosphate Ribose; Apoptosis; Area; Ataxia; Binding; Biochemical; Biological Models; Catabolism; Cell Differentiation process; Cell Proliferation; Cell Respiration; Cell physiology; Cells; Characteristics; Chronic; Cleaved cell; Complex; Cytoprotection; DNA; DNA Adducts; DNA Damage; DNA Ligases; DNA Ligation; DNA Repair; DNA Repair Enzymes; DNA Single Strand Break; DNA biosynthesis; DNA glycosylase; DNA strand break; DNA-Directed DNA Polymerase; Data; Defect; Disease; Down-Regulation; Esters; Eukaryotic Cell; Excision; Excision Repair; Exposure to; Family member; Genes; Genome; Genomics; Glutamates; Glycoside Hydrolases; Human; Hydrolysis; Hydroxyl Radical; Immune response; In Vitro; Inflammation; Inflammatory Response; Inherited; Ionizing radiation; Lead; Lesion; Ligase; Ligation; Limb structure; Link; Maintenance; Malignant Neoplasms; Metabolism; Molecular; Molecular Conformation; Mono-S; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Nucleic Acids; Nucleotides; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Play; Post-Translational Protein Processing; Predisposition; Process; Production; Protein Conformation; Protein Dynamics; Proteins; RNA; RNA Binding; Reaction; Research; Ribonucleotides; Ribose; Roentgen Rays; Role; S Phase; Saccharomycetales; Signal Transduction; Structure; Surgical incisions; Testing; Topoisomerase; Toxic Environmental Substances; Transcriptional Regulation; Wood material; Work; adduct; base; carboxyl group; computerized data processing; coping; cytotoxic; emergency service responder; environmental stressor; improved; in vivo; inorganic phosphate; molecular recognition; nervous system disorder; oculomotor; oxidative DNA damage; poly(ADP-ribose) protein complex; prevent; programs; protein folding; repaired; response; seal; structural biology

Our work focuses on two main project areas, i) DNA damage recognition and processing, and ii) DNA damage signaling. Progress in the last year in these areas is summarized below: i) DNA damage recognition and processing. Aptx is a conserved eukaryotic DNA repair enzyme that is important for protection of cells from oxidative DNA damage, and APTX mutations cause the hereditary neurodegenerative disorder Ataxia with Oculomotor Apraxia 1 (AOA1). In the ultimate step of DNA replication and repair processes, DNA ligases seal DNA nicks through with a mechanism that can abort when the ligase encounters damaged DNA. Such "abortive ligation" generates a secondary form of damage, 5'-adenylated DNA-termini, which is corrected by Aptx to protect genomic integrity. To understand the context for Aprataxin (Aptx) deadenylation repair we examined the importance of Aptx to RNaseH2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We demonstrated that DNA ligases generate adenylated 5′-ends containing a ribose characteristic of RNaseH2 incision. Aptx efficiently repairs adenylated RNA-DNA, and acting in an RNA-DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure-function studies of human Aptx/RNA-DNA/AMP/Zn complexes define a mechanism for detecting and reversing adenylation at RNA-DNA junctions. This involves A-form RNA-binding, proper protein folding and conformational changes, all of which are impacted by heritable APTX mutations in Ataxia with Oculomotor Apraxia 1 (AOA1). Together, these results suggest that accumulation of adenylated RNA-DNA may contribute to neurological disease. ii) DNA damage signaling. ADP-ribosylation is a reversible post-translational protein modification implicated in a range of cellular processes, including DNA repair, transcriptional regulation, cell differentiation and proliferation, inflammatory and immune responses, and apoptosis. PARPs use NAD+ as a substrate and covalently attach an ADP-ribose nucleotide, predominantly to the carboxyl group of glutamate residues on target proteins. Some PARP family members can subsequently add additional ADP-ribose units through glycosidic ribose-ribose bonds to generate a PAR chain specific hydrolysis of ribose-ribose bonds in PAR chains is catalysed by PAR glycohydrolase (PARG), but PARG is unable to cleave the ester bond between the ADP-ribose unit and the glutamate. We identified and structurally characterized an enzymatic activity in the human macrodomain containing protein C6orf130 that catalyses this step of PAR catabolism. We propose a cellular role for C6orf130 protein in the removal of the terminal ADP-ribose unit linked to PARP-modified proteins, by directly reversing protein mono(ADP-ribosyl)ation or by completing the reversal of protein poly(ADP-ribosyl)ation following the PARG reaction. Hence we have renamed this protein Terminal ADP-Ribose protein Glycohydrolase (TARG1). X-ray structures of C6orf130/TARG1 and biochemical data suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130/TARG1 protein from cells leads to proliferation and DNA repair defects, and homozygous mutation of the C6orf130 gene is found in patients with severe neurodegeneration. In ongoing studies, we are testing hypotheses that TARG1 activity plays roles in: 1) Catabolism of PAR chains in conjunction with PARG (poly-adp-ribose glycohydrolase) or, 2) Down-regulation of Parp1 signaling through direct reversal (removal of mono-ADP-ribose) of Parp1 and ADPR-modified proteins.

我们的工作主要集中在两个主要的项目领域，i) DNA损伤识别和处理，ii) DNA损伤信号。过去一年在这些领域取得的进展总结如下：