MECHANISMS OF GENOME INSTABILITY

基因组不稳定的机制

• 批准号: 6106746
• 负责人: MICHAEL A RESNICK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 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/6106746
• 关键词: DNA directed DNA polymerase; enzyme activity; frameshift mutation; genetic recombination; genome; human; human genetic material tag; nucleic acid repetitive sequence; tissue /cell culture

Summary of Work: The stability of genomes, particularly human, is influenced by opportunities for recombination between long (0.3 - 10 kb) repeats of diverged DNAs such as Alus and LINES, rearrangements between short (4-10 bp) random repeats and replication slippage within micro- and minisatellite DNAs. These repeats are abundant in the genome and represent at-risk motifs (ARMs), because of the high potential for genetic change. Long inverted repeats (IRs) can stimulate recombination and deletion. We found that the level of stimulation in yeast is directly related to the length of IRs and inversely related to the size of spacer between the IRs. A perfect 1 kb IR palindrome increased mitotic recombination approximately 20,000-fold. Large IRs can also stimulate recombination in mammalian cells, leading to novel recombinants. ARMs are especially unstable in the presence of some mutators, indicating that they can be tools for identifying mutators. For example, using pol3-t (DNA-Pol delta) and rad27 (Fen1) mutations that affect DNA replication, we demonstrated that minisatellites can be employed to detect mutators other than those arising from mismatch repair (MMR) defects. ARMs have also enabled us to detect weak mutators, which is important in considering human polymorphic variants that might have a subtle mutator effect or that might exhibit strong synergistic interactions. Using long homonucleotide runs we identified several weak mutators that could exhibit strong pairwise synergistic interactions including: i) dominant negative allele of the MMR gene MSH2; ii) the 5'-<3' exonuclease EXO1, that is potentially involved in MMR and shows strong mutator effects when combined with DNA Pol proofreading defects; iii) a novel mutator allele in pol2 (DNA Pol epsilon) that shows strong mutator effects when combined with an exo1-null. Long homonucleotide runs also proved useful for studying interactions of human MMR genes in yeast. Overexpressed hMSH2 and hMSH6 genes caused a strong mutator phenotype. The high sensitivity also enabled us to detect subtle mutator effects of overexpressed hMSH2 and hMSH3.

工作总结：基因组的稳定性， 尤其是人类，受到重组机会的影响 在不同DNA(如ALU)的长(0.3-10 kb)重复之间 和线条之间的重排，短(4-10个碱基对)随机 微小卫星内的重复和复制滑移 DNA。这些重复序列在基因组中大量存在，代表着 高危基序(ARM)，因为遗传的可能性很高 变化。长反向重复序列(IR)可以刺激重组 和删除。我们发现酵母中的刺激水平是 与IRS的长度直接相关，与大小成反比 国税局之间的间隔物。完美的1kb IR回文增加 有丝分裂重组约20,000倍。大型国税局可以 也刺激了哺乳动物细胞的重组，导致了新的 重组体。手臂在人的面前尤其不稳定 一些突变子，表明它们可以作为识别 变种人。例如，使用pol3-t(DNA-POL增量)和rad27 (Fen1)影响DNA复制的突变，我们证明了 小卫星可以用来检测那些以外的变种。 由失配修复(MMR)缺陷引起。武器也有 使我们能够检测到微弱的变种，这在 考虑到人类的多态变异可能具有微妙的 突变子效应或可能表现出很强的协同作用。 使用长时间的同源核苷酸序列，我们发现了几个弱的 可能表现出强烈的成对协同作用的变异体 包括：i)MMR基因MSH2的显性负等位基因；ii) 可能参与MMR的5‘-3’外切酶EXO1 当与DNA Pol结合时，显示出强烈的诱变效应 校对缺陷；iii)pol2(DNA Pol)中的一个新的突变等位基因 Epsilon)，当与一个 Exo1-空。长时间的同源核苷酸运行也被证明对 研究人MMR基因在酵母中的相互作用。 HMSH2和hMSH6基因的过表达导致了一个强大的突变体 表型。高灵敏度也使我们能够探测到微妙的 高表达hMSH2和hMSH3的诱变效应。