Structural Biology of Genome Maintenance and DNA repair

我们的工作集中在两个主要项目领域，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概述如下： 1）DNA损伤识别和处理。 APTX是一种保守的真核DNA修复酶，对保护细胞免受氧化DNA损伤很重要，APTX突变导致遗传性神经退行性疾病共济失调伴眼用失用1（AOA 1）。在DNA复制和修复过程的最后一步，DNA连接酶通过一种机制密封DNA切口，当连接酶遇到受损的DNA时，这种机制可以中止。这种“失败的连接”产生第二种形式的损伤，5 '-腺苷酸化的DNA末端，其被Aptx校正以保护基因组完整性。为了理解Aprataxin（Aptx）去腺苷化修复的背景，我们研究了Aptx对RNA酶H2依赖性切除修复（RER）的重要性，这种损伤经常被引入DNA，一种核糖核苷酸。我们证明了DNA连接酶产生腺苷酸化的5-末端，其含有RNaseH 2切割的核糖特征。Aptx有效地修复腺苷酸化的RNA-DNA，并在RNA-DNA损伤反应（RDDR）中起作用，促进细胞存活并防止经历RER的芽殖酵母中的S期检查点激活。人Aptx/RNA-DNA/AMP/Zn复合物的结构-功能研究定义了检测和逆转RNA-DNA连接处腺苷酸化的机制。这涉及A型RNA结合，适当的蛋白质折叠和构象变化，所有这些都受到共济失调伴眼用失用症1（AOA 1）中遗传性APTX突变的影响。总之，这些结果表明腺苷酸化RNA-DNA的积累可能有助于神经系统疾病。 ii）DNA损伤信号传导。 ADP-核糖基化是一种可逆的翻译后蛋白质修饰，涉及一系列细胞过程，包括DNA修复、转录调节、细胞分化和增殖、炎症和免疫反应以及细胞凋亡。PARP使用NAD+作为底物，并共价连接ADP-核糖核苷酸，主要是靶蛋白上谷氨酸残基的羧基。一些PARP家族成员随后可以通过糖苷核糖-核糖键添加额外的ADP-核糖单元以产生PAR链，PAR链中的核糖-核糖键的PAR链特异性水解由PAR糖水解酶（PARG）催化，但PARG不能切割ADP-核糖单元和谷氨酸之间的酯键。我们确定和结构特征的酶活性在人的宏域含有蛋白质C6 orf 130，催化这一步的PAR catalysts。我们提出了C6 orf 130蛋白在去除连接到PARP修饰的蛋白的末端ADP-核糖单元中的细胞作用，通过直接逆转蛋白单（ADP-核糖基）化或通过完成PARG反应后蛋白聚（ADP-核糖基）化的逆转。因此，我们将这种蛋白质重命名为末端ADP-核糖蛋白糖水解酶（TARG 1）。C6 orf 130/TARG 1的X射线结构和生化数据表明，涉及一个短暂的C6 orf 130赖氨酰（ADP-核糖）中间体的催化逆转机制。此外，C6 orf 130/TARG 1蛋白从细胞中的缺失导致增殖和DNA修复缺陷，并且在患有严重神经变性的患者中发现C6 orf 130基因的纯合突变。在正在进行的研究中，我们正在测试TARG 1活性在以下方面发挥作用的假设：1）与PARG（聚ADP核糖糖水解酶）一起分解代谢PAR链，或2）通过直接逆转（去除单ADP核糖）下调Parp 1信号传导Parp 1和ADPR修饰的蛋白质。

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.