Chemistry and Biology of DNA Ligation

DNA 连接的化学和生物学

基本信息

批准号：
10302270
负责人：
Patrick J O'Brien
金额：
$ 29.86万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-12-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10302270
关键词：
APTX gene Antibiotics Binding Biochemical Biological Biological Assay Biology Cell Nucleus Cell physiology Cells Chemistry Clinical Collaborations Common Variable Immunodeficiency Coupled Crystallization DNA DNA Binding DNA Damage DNA Ligases DNA Ligation DNA Primase DNA Repair DNA Repair Pathway DNA Sequence Alteration DNA biosynthesis DNA ligase I DNA-Directed DNA Polymerase Defect Development Diagnosis Discrimination Disease Disease model Doctor of Philosophy Enzymatic Biochemistry Enzymes Fingers Foundations Genetic Goals Grant Growth Health Human Immune Immunology Inherited Institutes Kinetics Knowledge LIG4 gene Learning Ligase Ligation Maintenance Malignant Neoplasms Mammalian Cell Metals Michigan Mitochondria Mitochondrial DNA Modeling Modification Molecular Mus Mutation Neuroblastoma Nonhomologous DNA End Joining Normal Cell Nucleotides Organism Pathway interactions Patients Pediatric Hospitals Phenylalanine Polymerase Property Protein Isoforms Proteins Recombinants Research Ribonucleotides Role Severe Combined Immunodeficiency Side Site Site-Directed Mutagenesis Specificity Structure Structure-Activity Relationship Syndrome Testing Thermodynamics Translating Universities Work X-Ray Crystallography XRCC1 gene cancer therapy cofactor congenital immunodeficiency defined contribution disorder risk experimental study human DNA human disease inhibitor insight magnesium ion mouse development mutant novel novel therapeutic intervention overexpression repaired response sugar

项目摘要

The goal of this proposal is to determine the mechanism and specificity of human DNA ligases. All organisms have an absolute requirement for DNA replication and DNA repair in order to synthesize new cells and to maintain correct cellular functions. DNA ligases catalyze the ultimate step in DNA replication and most DNA repair pathways, however most research has focused on DNA polymerases and therefore we lack a fundamental understanding of DNA ligase mechanisms and specificities. It is essential to learn the molecular mechanism of human DNA ligases and that these insights will have significant value for understanding human health. Our work is guided by the known biology of DNA repair and replication and we focus on unanswered puzzles that cannot be explained by current understanding of these pathways. Some recent clinical examples of where this knowledge is critical is in patients with inherited mutations in DNA ligase genes, such as LIG1 syndrome which causes a Primary ImmunoDeficiency and LIG4 syndrome which causes Severe Combined ImmunoDeficiency (SCID), and in abnormal states such as cancer in which ligases, especially LIG3, have been overexpressed. We will use quantitative mechanistic enzymology and x-ray crystallography (in collaboration with Dr. Scott Williams) to characterize human DNA ligase 1 (LIG1) and DNA ligase 3 (LIG3). Genetic observations suggest that these enzymes have unique functions, but share some similarities. For example, LIG1 and LIG3 both function in DNA repair and replication in the nucleus, but only LIG3 functions in the mitochondria. We will extend our previous kinetic and structural studies of LIG1 to understand specificity of this enzyme and to define the minimal steps in locating and engaging a single strand break. We have recently succeeded in producing large quantities of recombinant LIG3 alpha and beta isoforms and we will perform a kinetic and thermodynamic characterization to understand similarities and differences with LIG1. For both enzymes, we will use site-directed mutagenesis to target specific functions, such as metal cofactor binding, DNA binding, and catalytic specificity. Analysis of these mutant proteins will provide an understanding of the molecular features of eukaryotic DNA ligation that will be invaluable to understand ligase function in normal cells and in human disease. The core objectives of the grant are to develop a molecular understanding of the mechanism and specificity of LIG1 and LIG3, but the mechanistic models that emerge will then be tested in an appropriate cell. This work will uncover the interconnections between different DNA repair pathways and provide a strong foundation for understanding regulatory interactions and modifications.

该提案的目标是确定人类 DNA 连接酶的机制和特异性。所有生物体对 DNA 复制和 DNA 修复有绝对的要求，以合成新细胞并维持正确的细胞功能。 DNA 连接酶催化 DNA 复制和大多数 DNA 的最终步骤修复途径，然而大多数研究都集中在 DNA 聚合酶上，因此我们缺乏对 DNA 连接酶机制和特异性的基本了解。学习分子生物学很重要人类 DNA 连接酶的机制，这些见解对于理解人类具有重要价值健康。我们的工作以已知的 DNA 修复和复制生物学为指导，我们专注于尚未解答的问题目前对这些途径的理解无法解释这些难题。最近的一些临床例子这一知识的关键在于 DNA 连接酶基因（例如 LIG1）具有遗传性突变的患者导致原发性免疫缺陷的综合征和导致严重联合的 LIG4 综合征免疫缺陷 (SCID) 以及癌症等异常状态，其中连接酶（尤其是 LIG3）具有被过度表达了。我们将使用定量机械酶学和 X 射线晶体学（在与 Scott Williams 博士合作）表征人类 DNA 连接酶 1 (LIG1) 和 DNA 连接酶 3 (LIG3)。遗传观察表明这些酶具有独特的功能，但也有一些相似之处。为了例如，LIG1和LIG3都在细胞核中的DNA修复和复制中发挥作用，但只有LIG3在细胞核中发挥作用。线粒体。我们将扩展之前对 LIG1 的动力学和结构研究，以了解这种酶并定义定位和接合单链断裂的最少步骤。我们最近有成功生产大量重组 LIG3 α 和 β 亚型，我们将进行动力学和热力学表征，以了解与 LIG1 的异同。对于两者酶，我们将使用定点诱变来靶向特定功能，例如金属辅因子结合， DNA 结合和催化特异性。对这些突变蛋白的分析将有助于了解真核 DNA 连接的分子特征对于理解正常连接酶的功能非常有价值细胞和人类疾病。该赠款的核心目标是发展对分子的理解 LIG1 和 LIG3 的机制和特异性，但出现的机制模型将在合适的细胞。这项工作将揭示不同 DNA 修复途径和为理解监管相互作用和修改提供坚实的基础。