RECOMBINATION AND DNA DIVERGENCE

重组和 DNA 分歧

• 批准号: 6106569
• 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/6106569
• 关键词: DNA; DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; Saccharomyces cerevisiae; bacterial genetics; biochemical evolution; enzyme activity; fungal genetics; genetic recombination; genome; nucleic acid repetitive sequence

Summary of Work: An important class of at-risk motifs (ARMs) that can lead to human genome instability involves interspersed repetitive sequences. These can be a source of large chromosomal rearrangements in somatic and germ cells. We are investigating mechanisms of recombination between repeat ARMs, impact of divergence, organization and size of repeats, environmental agent effects as well as genetic controls in the yeast Saccharomyces cerevisiae. This organism serves as a model genetic system for changes in human cells. The most abundant class of interspersed repeats in the human genome, Alus, comprise approximately 10% of total DNA. The average sequence divergence is 85% which might be a barrier to rearrangement. We developed a novel system in yeast to address the ability of homologous and diverged Alus that are organized in motifs present in the human genome to stimulate recombination. Of particular interest is the inverted Alu repeat motif which is found in the human genome, since we have shown that a palindrome formed between 1.0 kb URA3 genes can increase recombination approximately 20,000-fold. To investigate i) the potential for inverted Alu-repeats to stimulate genome instability and ii) the effect of sequence divergence, we constructed a set of quasipalindrome inserts (12 bp between the Alu repeats) with varying levels of identity. The 300 bp inverted Alu repeats with greater than 99% identity increased recombination nearly 1000-fold over that of either direct repeats or unique DNA. 95%, 85% and 76% identical IRs exhibited 100-, 5- fold and no increase, respectively. Surprisingly, mismatch repair defects do not affect recombination stimulation. Thus, even diverged inverted Alus can induce genetic rearrangements. This model system is being used to investigate the many factors that could contribute to repeat stimulated instability including mismatch and recombinational repair, replication and environmental agents. To quantitatively assess repeat ARMs in the human genome, we are analyzing the human genome data base for the presence of closely space Alu repeats. This will be extended to other repeats.

工作总结：一个重要的风险类别 可导致人类基因组不稳定性的ARMs包括 散布的重复序列。这可能是一个巨大的来源。 体细胞和生殖细胞中的染色体重排。我们 研究重复ARM之间的重组机制， 分歧的影响，重复的组织和大小， 环境因子的影响以及酵母中的遗传控制 酿酒酵母这种生物体作为一种遗传模型 人类细胞的变化。最丰富的一类 人类基因组中的散布重复序列，Alus，包括 大约10%的DNA。平均序列 发散度为85%，这可能是重排的障碍。我们 在酵母中开发了一种新的系统， 同源和分歧的Alus组织在基序存在 来刺激重组特别 感兴趣的是在人类中发现的反向Alu重复基序， 基因组，因为我们已经表明，回文形成之间 1.0 kb的URA 3基因可以增加重组， 两万倍研究i）反向重复序列的可能性 刺激基因组的不稳定性和ii）序列的影响 分歧，我们构建了一组准回文插入片段（12 bp 在Alu重复之间）具有不同的同一性水平。300 bp 具有大于99%同一性的反向Alu重复增加 重组近1000倍，无论是直接重复或 独特的DNA 95%、85%和76%相同的IR显示100-、5- 分别增加和不增加。令人惊讶的是，错配修复 缺陷不影响复合刺激。因此即使 分叉的倒向Alus可以诱导遗传重排。这 模型系统被用来研究许多因素， 可能导致重复刺激不稳定性，包括失配 以及重组修复、复制和环境因子。 为了定量评估人类基因组中的重复ARM，我们 分析人类基因组数据库， 空格Alu重复。这将扩展到其他重复。