Mammalian L1 retrotransposons as genetic characters

哺乳动物 L1 逆转录转座子作为遗传特征

• 批准号: 10253731
• 负责人: ANTHONY V. FURANO
• 金额: $ 60.69万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 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/10253731
• 关键词: Aging; Base Excision Repairs; Biological Sciences; Breast Cancer Cell; Breast Cancer cell line; DNA; DNA Damage; DNA Repair; DNA glycosylase; Deaminase; Diffuse; Disease; Encyclopedias; Enzymes; Episome; Excision; Family; Fossils; Frequencies; Genetic; Genets; Genome; Genomic DNA; Human; In Vitro; Induced Mutation; L1 Elements; Lyase; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mismatch Repair; Modernization; Mutagenesis; Mutation; Nature; Oxidative Stress; Oxides; Oxygen; Parasites; Pathway interactions; Phenotype; Polymerase; Process; Proteins; Publishing; RNA; Retrotransposon; Simian virus 40; Single-Stranded DNA; Site; Son; Source; Stress; System; Time; Transcript; base; cancer cell; in vivo; novel; repaired; sensor

MECHANISMS OF DNA REPAIR INDUCED MUTAGENESIS. We had implemented an experimental system to determine in vivo whether DNA repair can induce mutations in DNA that flanked the site of DNA repair and found that it does. In particular, the repair intermediates generated from repairing preformed normally occurring DNA mispairs that we introduced into an SV40-based episome were vulnerable at a low but statistically significant frequency to an APOBEC3 (A3) C-deaminase-mediated error-prone process. Both base excision repair (BER) and mismatch repair pathways (MMR) were involved in generating the obligatory single stranded substrate for TpC-preferring A3B deaminase. This process produced mutations similar to those typical of the mutator phenotypes in various cancers. The mechanistic basis of the mutator phenotype is not known but our studies suggested that normally error-free DNA repair processes can act as mutators providing a heretofore unexpected source of genetic changes that underlie disease, aging and evolutionary change. We applied our episome mutation sensor to pairs of established breast cancer cell lines that contain similar A3B levels and found that some pairs differed dramatically in mutagenic repair. We found that this difference was due to elevated expression of the bifunctional DNA glycosylase, NEIL2, which sensitizes breast cancer cells to both A3B-mediated mutations and double strand breaks (DSBs) by perturbing canonical BER. NEIL2 is normally involved in the removal of oxidized bases, which can accumulate under conditions of oxidative stress that often occurs in cancer cells. We showed that NEIL2 usurps the canonical BER DNA lyase, APE1, at abasic sites in a purified BER system, and that the nicked NEIL2 product is a poor substrate for the next enzyme in the BER pathway, beta polymerase. However, the nicked NEIL2 product can serve as an entry site for the MMR enzyme Exo1 in vitro to generate single-stranded DNA, which would be susceptible to both A3B and DSBs. Our findings that NEIL2 or Exo1 depletion mitigates the DNA damage caused by A3B expression, indicates that aberrant NEIL2 expression can abort normal BER and drive its intermediates into an MMR pathway to generate A3 substrates. In vivo evidence for the involvement of MMR in A3-mediated mutations has just been published (Mas-Ponte & Supek: DNA mismatch repair promotes APOBEC3-mediated diffuse hypermutation in human cancers. Nature Genet. 2020). We are now using mass spectrometry to profile NEIL2-interacting proteins under conditions of oxygen stress.

DNA修复诱导突变的机制。我们已经实施了一个实验系统，以确定体内DNA修复是否可以诱导DNA修复位点两侧的DNA突变，并发现确实如此。特别是，通过修复预先形成的正常发生的DNA错对而产生的修复中间体，我们将其引入到基于sv40的片段中，在APOBEC3 (A3) c -脱氨酶介导的易出错过程中，修复中间体的频率很低，但具有统计学意义。碱基切除修复（BER）和错配修复途径（MMR）都参与了tpc偏好的A3B脱氨酶产生必需的单链底物。这个过程产生的突变与各种癌症中典型的突变表型相似。突变表型的机制基础尚不清楚，但我们的研究表明，正常情况下无错误的DNA修复过程可以作为突变体，为疾病、衰老和进化变化提供迄今为止意想不到的遗传变化来源。我们将我们的片段突变传感器应用于含有相似A3B水平的已建立的乳腺癌细胞系对，发现一些对在诱变修复方面存在显着差异。我们发现这种差异是由于双功能DNA糖基酶NEIL2的表达升高，NEIL2通过干扰标准BER使乳腺癌细胞对a3b介导的突变和双链断裂（DSBs）敏感。NEIL2通常参与氧化碱基的去除，氧化碱基可以在癌细胞中经常发生的氧化应激条件下积累。我们发现，在纯化的BER系统中，NEIL2在基本位点篡位了典型的BER DNA裂解酶APE1，并且缺口的NEIL2产物是BER途径中下一个酶-聚合酶的差底物。然而，缺口的NEIL2产物可以作为MMR酶Exo1在体外产生单链DNA的进入位点，而单链DNA对A3B和dsb都很敏感。我们发现NEIL2或Exo1缺失减轻了A3B表达引起的DNA损伤，这表明异常的NEIL2表达可以中止正常的BER并驱动其中间体进入MMR途径生成A3底物。MMR参与a3介导突变的体内证据刚刚发表（Mas-Ponte & Supek: DNA错配修复促进人类癌症中apobec3介导的弥漫性超突变）。自然基因。2020)。我们现在正在使用质谱分析在氧胁迫条件下neil2相互作用的蛋白质。