Structural Biology of Genome Maintenance and DNA repair

我们的工作主要集中在两个项目领域，i)DNA损伤识别和处理，以及ii)DNA损伤信号。以下是过去一年在这些领域取得的进展： I)DNA损伤识别和处理。 AptX是一种保守的真核细胞DNA修复酶，对保护细胞免受DNA氧化损伤具有重要作用，而aptX突变可导致遗传性神经退行性疾病共济失调伴动眼失用症1(AOA1)。在DNA复制和修复过程的最终步骤中，DNA连接酶通过一种机制封闭DNA缺口，当连接酶遇到受损的DNA时，该机制可以终止。这种“流产的连接”会产生第二种形式的损伤，即5‘-腺化DNA末端，aptX会纠正这种末端，以保护基因组的完整性。为了了解Aprataxin(AptX)死烯基化修复的背景，我们研究了aptX对RNaseH2依赖的切除修复(RER)的重要性，这种损伤经常被引入DNA，一种核苷酸。我们证明，DNA连接酶产生腺化的5-末端，含有RNaseH2切口的核糖特征。APTX有效地修复腺化的RNADNA，并作用于RNADNA损伤反应(RDDR)，促进细胞存活，并阻止正在进行RNADNA损伤反应的芽酵母中S阶段的检查点激活。人aptX/RNA-DNA/AMP/Zn复合体的结构-功能研究为检测和逆转RNA-DNA连接处的腺化提供了一种机制。这涉及A型RNA结合、正确的蛋白质折叠和构象变化，所有这些都受到患有动眼失用症1(AOA1)的共济失调患者可遗传的aptX突变的影响。综上所述，这些结果表明，腺化RNA-DNA的积累可能导致神经疾病。 Ii)DNA损伤信号。 ADP-核糖化是一种可逆的翻译后蛋白质修饰，参与一系列细胞过程，包括DNA修复、转录调控、细胞分化和增殖、炎症和免疫反应以及细胞凋亡。PAPS以NAD为底物，共价连接ADP-核糖核苷酸，主要连接到靶蛋白上谷氨酸残基的羧基上。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-核糖单元来生成PAR链。PAR链中核糖-核糖键的特异性水解是由PAR糖水解酶(PARG)催化的，但PARG不能切割ADP-核糖单元和谷氨酸之间的酯键。我们鉴定并结构表征了人类大域蛋白C6orf130中的一种酶活性，该蛋白催化PAR分解代谢的这一步骤。我们提出了C6orf130蛋白在移除连接到PARP修饰蛋白的末端ADP-核糖单元中的细胞作用，方法是直接逆转蛋白质单(ADP-核糖基)化或完成PARG反应后蛋白质聚(ADP-核糖基)化的逆转。因此，我们将该蛋白重命名为末端ADP-核糖蛋白糖水解酶(TARG1)。C6orf130/TARG1的X射线结构和生化数据表明，C6orf130赖氨酰-(ADP-核糖)中间体参与了催化逆转的机制。此外，C6orf130/TARG1蛋白从细胞中耗尽会导致增殖和DNA修复缺陷，在严重的神经变性患者中发现C6orf130基因的纯合子突变。在正在进行的研究中，我们正在验证TARG1活性在以下几个方面发挥作用的假设：1)PAR链与PARG(聚-ADP-核糖水解酶)一起分解代谢，或2)通过直接逆转PARP1和ADPR修饰的蛋白质而下调PARP1信号。

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.