DNA Replication, Repair, and Mutagenesis In Eukaryotic And Prokaryotic Cells

真核和原核细胞中的 DNA 复制、修复和诱变

基本信息

批准号：
8351143
负责人：
ROGER WOODGATE
金额：
$ 214.31万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
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/8351143
关键词：
Acids Archaea Archaeology B-Lymphocytes Bacteria Biological Assay Biology Clinical Complex Cytosine DNA DNA Damage DNA Sequence DNA Shuffling DNA biosynthesis DNA lesion DNA-Directed DNA Polymerase Data Deamination Detection Diagnostic Digestion Dust Elements Employee Strikes Engineering Enzymes Eukaryota Eukaryotic Cell Event Family Forensic Medicine Genomics Human Immunoglobulin Genes Laboratories Life Medicine Methods Modeling Molecular Molecular Genetics Mutagenesis Mutation Organism Orthologous Gene Pathway interactions Peat Polymerase Polymerase Chain Reaction Process Prokaryotic Cells RNA Recovery Resistance Scientist Soil Specimen Tars Thermus Thermus thermophilus United States National Institutes of Health Uracil activation-induced cytidine deaminase base bone clay endonuclease in vivo inhibitor/antagonist novel prognostic repaired uracil-DNA glycosylase

项目摘要

Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into DNA. These studies span the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes In humans, Activation-Induced Deaminase (AID) initiates diversity of immunoglobulin genes through deamination of Cytosine to Uracil. However, it has yet to be conclusively determined if the deamination event occurs in DNA, or RNA. Although most data support DNA deamination, there been is no physical evidence of Uracil residues in immunoglobulin genes. In a collaborative study with Patricia Gearhart (NIA/NIH), we demonstrated their presence by determining the sensitivity of DNA to digestion with Uracil DNA glycosylase (UNG) and abasic endonuclease. Using several different methods of detection, we identified Uracil residues in the variable and switch regions of human immunoglobulin genes. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly Cytosine bases. Our data therefore provides the first direct evidence supporting the model that AID functions by deaminating Cytosine residues in DNA, rather than in RNA. DNA polymerases from Archaea are often thermostable and have been used for several decades in the polymerase chain reaction (PCR). PCR enables the detection, amplification and interrogation of DNA sequences from minute starting quantities, down to single DNA molecules. This has enabled a wealth of applications in medicine and biology ranging from clinical diagnostics, prognostics, and forensics, to molecular genetics including molecular archaeology and palaeobiology. However, the utility of PCR assays and the recovery of amplicons from such specimens can be greatly hindered, or even abrogated, by the presence of potent inhibitors. In a collaborative study with Phillip Holliger (MRC, Cambridge UK), we used molecular breeding and compartmentalized self-replication (CSR) of eight different Thermus DNA polymerase orthologs to engineer novel DNA polymerases with a broad resistance to complex environmental inhibitors. One such enzyme, called 2D9, was a chimeric polymerase comprising sequence elements derived from DNA polymerases from Thermus aquaticus, Thermus oshimai, Thermus thermophilus and Thermus brockianus. Remarkably, the 2D9 polymerase displayed a striking resistance to a broad spectrum of complex inhibitors of highly divergent composition including humic acid, bone dust, coprolite, peat extract, clay-rich soil, cave sediment and tar. We believe that 2D9 chimeric polymerase promises to have utility in PCR-based applications in a wide range of scientific fields including palaeobiology, archaeology, conservation biology, forensic and historic medicine.

基因组完整性实验室（LGI）中的科学家研究了将突变引入DNA的机制。这些研究跨越了进化光谱，包括细菌，古细菌和真核生物的研究在人类中，激活诱导的脱氨酶（AID）通过将胞嘧啶对尿嘧啶的脱氨酸启动免疫球蛋白基因的多样性。但是，尚未确定脱氨酸事件是否发生在DNA或RNA中。尽管大多数数据支持DNA脱氨基，但没有物理证据表明免疫球蛋白基因中的尿嘧啶残基。在与Patricia Gearhart（NIA/NIH）的一项合作研究中，我们通过确定DNA对使用尿嘧啶DNA糖基酶（UNG）和阿无核核酸内切酶消化的敏感性来证明它们的存在。使用几种不同的检测方法，我们确定了人类免疫球蛋白基因的变量和开关区域中的尿嘧啶残基。尿嘧啶残基在B细胞刺激的24小时内产生，两种DNA链都存在，并被发现主要取代了胞嘧啶碱基。因此，我们的数据提供了第一个直接证据，支持该模型通过脱离DNA中的胞嘧啶残基而不是RNA中的胞嘧啶残基来帮助功能。来自古细菌的DNA聚合酶通常是热稳定的，并且在聚合酶链反应（PCR）中已使用了几十年。 PCR可以从微小起始量到单个DNA分子对DNA序列的检测，扩增和询问。这使得从临床诊断，预后和法医学到包括分子考古学和古生物学在内的分子遗传学，都可以在医学和生物学上进行丰富的应用。但是，通过存在有效的抑制剂，PCR分析的实用性和从此类标本中恢复的扩增子可以极大地阻碍，甚至消除。在与Phillip Holliger（MRC，Cambridge UK）的合作研究中，我们使用了八种不同的Thermus DNA聚合酶直系同源物的分子育种和分室化自我复制（CSR），对新颖的DNA聚合酶具有对复杂环境抑制剂的广泛抗性。一种称为2d9的酶是一种嵌合聚合酶，其中包括源自Thermus aquateus，Thermus Oshimai，Thermus Thermus Thermophilus和Thermus Brockianus的DNA聚合酶的序列元件。值得注意的是，2D9聚合酶表现出对高度发散组成的复杂抑制剂的抗抗药性，包括腐殖酸，骨粉，coprolite，泥炭提取物，泥炭提取物，富含粘土的土壤，洞穴沉积物和焦油。我们认为，2D9嵌合聚合酶有望在基于PCR的应用中在广泛的科学领域中具有实用性，包括古生物学，考古学，保护生物学，法医和历史医学。