一种新型的遗传不稳定现象-短片段DNA序列重复的分子机制的探究

Genetic mutations in essence are changes in DNA sequences, that are inheritable and are usually irreversible. Reversible inheritable changes, on the other hand, may be uniquely beneficial under certain circumstances such as flexible adaptation to environment fluctuation. .Rapamycin is a small molecule inhibitor targeting the protein kinase TORC. In fission yeast, combined treatment of caffeine and rapamycin (called rapamycin treatment in short thereafter) is required for complete blockage of cell growth. Thirty-three gene deletions have been identified to cause stable rapamycin resistance. .We set out to explore unstable inheritable mechanisms using unstable drug resistance as the experimental tool. We have isolated fourteen new fission yeast strains that are resistant to rapamycin. In contrast to the identified gene deletion mutants, the drug resistance of these fourteen strains is unstably inherited. Through whole-genome sequence, we identified a novel form of genetic instability that we called short DNA segment duplication (SSD). In eight of the fourteen (~60%) strains, three short DNA segments within ssp1 gene were identified, each with 5-8bp microhomology at the ends, individually underwent tandem duplication in one of the strains, causing inactivation of ssp1 gene and resistance to rapamycin. Precise reversal of the duplication back to the original sequence occurs spontaneously at a high frequency (1/10^4), that causes reactivation of ssp1 and loss of drug resistance. .In this study, we aim at delineating the molecular mechanism of SSD. We propose to identify features in DNA sequence as well as gene function of ssp1 that render it to be the hotspot of SSD. In addition, we will explore the possible relationship of microcohomogy-mediated short segment duplication with DNA replication press and specific DNA damage repair pathways. We also propose to identify novel genes that are functionally implicated in SSD. The success of this project will systematically and comprehensively reveal a new mechanism of genome instability of reversible, microhomology-mediated genetic rearrangement.

DNA序列的突变通常是稳定遗传、不可逆的。而可逆的遗传变化可能对生物体有独特的效益，如更灵活地适应环境的波动。.雷帕霉素通过抑制TORC蛋白激酶抑制细胞生长。已知在裂殖酵母中多个基因敲除导致稳定的雷帕霉素抗性。.我们试图以不稳定抗药性为切入点，发现新的不稳定遗传机制。我们筛选得到了14个不稳定抗药酵母菌株。通过全基因组测序，我们发现一种新型的遗传不稳定现象 - DNA短片段重复 (Short Segment Duplication，SSD)。在蛋白激酶 (CamKK/ssp1)基因中，我们发现三段DNA片段分别在8个不稳定抗药株中发生重复使ssp1失活，导致抗药性。DNA片段重复可精确回复使失活的ssp1复活，导致抗药性不稳定。这些DNA片段的两端都具有微型同源臂（5-8bp）。.本研究旨在探究SSD现象的发生与回复的分子机制，着重阐明ssp1作为SSD热点的DNA序列及基因功能特征。我们将探究SSD的发生与DNA复制压力和 DNA损伤修复的可能关系，并系统地筛选参与SSD回复的基因。本课题的成功将首次确定一种全新的可逆的基因重排现象并阐明其分子机制。

大部分遗传改变都存在一定的回复概率，一方面这些改变可能对某些不利的生长环境具有一定的抵抗作用，如：药物处理条件，但在不利条件解除后，这些改变的存在可能对生物体的正常生长起阻碍作用。因此，如何对多变的外界环境做出快速的遗传改变从而适应环境，对生物体而言是一项重要的挑战。我们的研究在裂殖酵母中发现了一种快速且可逆的抗药性突变---微型同源臂介导的串联重复（简称为MTD）,在一定的条件下能够回复至野生型序列。通过对10,000x全基因组测序结果进行MTD分析，我们在单细胞起源的细胞群体中发现了近6000个亚克隆MTDs突变位点；MTD的频率受到发生概率、回复概率、及适应性等方面共同作用，通过使用特殊的模拟模型及高温筛选策略，我们初步了解了MTD的发生频率是如何受调控的。此外，我们对裂殖酵母所有的微型同源臂序列（MHPs）进行了注释，发现MHP序列在基因组上普遍存在（~2.5×107个），而模型预测的发生MTD概率最高的MHP位点往往插入MTDs后不会改变基因的阅读框，因此基因组上MHPs的存在及MTDs的发生与恢复对维持编码序列的演化起重要的作用。我们的研究揭示了一种普遍且可逆的遗传变化的机制，这种遗传改变对生物体快速应对环境波动、促进基因组进化多样性起重要作用。