Crystallographic studies of macromolecular structures

大分子结构的晶体学研究

基本信息

批准号：
10008871
负责人：
Lars Pedersen
金额：
$ 128.97万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
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/10008871
关键词：
8-hydroxyguanosine ATP phosphohydrolase ATPase Domain Active Sites Adenine Adenosine Amino Acids Anabolism Behavior Bypass Chondroitin Sulfates Chromosomes Communities Crystallization Cytidine DNA DNA Double Strand Break DNA Repair DNA Structure DNA biosynthesis DNA polymerase mu Dimerization Facioscapulohumeral Muscular Dystrophy Family Gene Silencing Genomic Imprinting Goals Health Human Hydroxyl Radical Immunoglobulin Genes LIG4 gene Ligase Ligation Macromolecular Complexes Maintenance Missense Mutation Molecular Conformation Molecular Structure National Institute of Environmental Health Sciences Nonhomologous DNA End Joining Nuclear Receptors Nucleotides Pathway interactions Polymerase Protein Analysis Proteins Publishing Reporting Research Research Support Ribonucleotides Roentgen Rays Role Structural Protein Structure Techniques Testing Transducers Ubiquitin Variant Work X Inactivation alpha synuclein biological systems dimer flexibility genotoxicity improved inorganic phosphate insight interest mutant novel phosphodiester protein complex protein function protein structure repaired seal

项目摘要

Our lab works on a variety of different biological systems including DNA replication/repair, nuclear receptors, and heparan/chondroitin sulfate biosynthesis. Listed below are some notable results from our research in this past year: 1) SMCHD1 (structural maintenance of chromosome flexible hinge domain containing 1) is a 2005 amino acid protein involved in gene silencing, with reported roles in X inactivation, genomic imprinting, and non-homologous end joining (NHEJ). Missense mutations in SMCHD1 are associated with arhinia, and Facioscapulohumeral Muscular Dystrophy Type 2 (FSHD2). Last year, we solved the initial X-ray crystal structure of the SMCHD1 ATPase module (aa 24-580) ,and this year focused on structural refinement and analysis of the protein. The ATPase module is composed of three domains: ubiquitin-like domain (UBL), ATPase domain, and transducer domain. We determined that while both the ATPase and transducer domains are required for ATPase activity, the UBL domain is not. However, the UBL is required for dimerization, consistent with the domain-swap observed in the crystal structure dimer. We also characterized 11 variants associated with arhinia and FSHD2. Most of the mutants maintained near wild-type activity and all of the arhinia mutants dimerized. Interestingly, dimerization was greatly diminished in the FSHD2 variants tested. Most notably, the variant Y283C showed a slight increase in ATPase activity even in the absence of dimerization. 2) Classical non-homologous end-joining (NHEJ) is the pathway through which DNA double strand breaks, resulting from immunoglobulin gene maturation or genotoxic DNA damage, are repaired. Human X family polymerase Mu is involved in faithful repair of these breaks by incorporating correctly paired nucleotides to fill gaps in the broken DNA, which are subsequently sealed by DNA ligase IV (LigIV). LigIV utilizes ATP to catalyze the ligation step between the 3-hydroxyl and the 5-phosphate of the break to generate a new phosphodiester bond. In support of research carried out in Dr. Thomas Kunkels Replication Fidelity Group, our lab published novel structures of DNA polymerase Mu engaged with DNA substrates containing an 8-oxoG in the template strand to better understand how Pol Mu bypasses this type of damage using both deoxyribo- and ribonucleotides. Our work demonstrates that Pol Mu prefers to misinsert adenine rather than correctly paired cytidine nucleotides opposite the template 8-oxoG, which suggests that, given cellular nucleotide concentrations, the most likely nucleotide inserted would be a ribo- rather than deoxyriboadenosine. This is consistent with our crystal structures, which demonstrate that adenosine is easily accommodated opposite a syn 8-oxoG with no conformational distortion within the active site.

我们的实验室致力于各种不同的生物系统，包括 DNA 复制/修复、核受体和乙酰肝素/硫酸软骨素生物合成。下面列出了我们去年研究的一些值得注意的结果： 1) SMCHD1（包含 1 的染色体柔性铰链结构域的结构维护）是一种 2005 年氨基酸蛋白，参与基因沉默，据报道在 X 失活、基因组印记和非同源末端连接 (NHEJ) 中发挥作用。 SMCHD1 的错义突变与 Arhinia 和 2 型面肩肱型肌营养不良症 (FSHD2) 相关。去年，我们解决了SMCHD1 ATPase模块（aa 24-580）的初步X射线晶体结构，今年重点关注蛋白质的结构精修和分析。 ATPase 模块由三个结构域组成：泛素样结构域 (UBL)、ATPase 结构域和转导结构域。我们确定，虽然 ATP 酶和转导结构域都是 ATP 酶活性所必需的，但 UBL 结构域不是。然而，UBL 是二聚化所必需的，这与在晶体结构二聚体中观察到的域交换一致。我们还鉴定了与 arhinia 和 FSHD2 相关的 11 种变异。大多数突变体保持接近野生型的活性并且所有arhinia突变体二聚化。有趣的是，在测试的 FSHD2 变体中，二聚化大大减少。最值得注意的是，即使在没有二聚化的情况下，变体 Y283C 的 ATP 酶活性也略有增加。 2) 经典的非同源末端连接 (NHEJ) 是修复由免疫球蛋白基因成熟或基因毒性 DNA 损伤引起的 DNA 双链断裂的途径。人类 X 家族聚合酶 Mu 通过掺入正确配对的核苷酸来填充断裂 DNA 中的间隙，从而参与对这些断裂的忠实修复，随后由 DNA 连接酶 IV (LigIV) 密封这些间隙。 LigIV 利用 ATP 催化断裂的 3-羟基和 5-磷酸之间的连接步骤，生成新的磷酸二酯键。为了支持 Thomas Kunkels 复制保真集团博士进行的研究，我们的实验室发表了 DNA 聚合酶 Mu 的新颖结构，该结构与模板链中含有 8-oxoG 的 DNA 底物结合，以更好地了解 Pol Mu 如何利用这两种方法绕过此类损伤脱氧核糖核苷酸和核糖核苷酸。我们的工作表明，Pol Mu 更喜欢错误插入腺嘌呤，而不是与模板 8-oxoG 相对的正确配对的胞苷核苷酸，这表明，给定细胞核苷酸浓度，最有可能插入的核苷酸是核糖而不是脱氧核糖腺苷。这与我们的晶体结构一致，表明腺苷很容易与 syn 8-oxoG 相对，在活性位点内没有构象扭曲。