Identification of distinct pathways for DSB formation at palindromic repeats

采用发夹和十字形结构的回文序列是染色体突变的有效来源。 断裂和重排，并在疾病的发病机制中发挥重要作用。在人类中，对于 例如，在非复发性和复发性的染色体断裂点上发现了回文 可能导致伊曼纽尔综合征的基因易位此外，回文介导的染色体 基因重排引起几种类型的εγδβ地中海贫血、X连锁先天性肥大综合征和 遗传性肾细胞癌最后，回文酶与基因的扩增有关， 结肠癌和乳腺癌、髓母细胞瘤和淋巴瘤的肿瘤发生。尽管受到了 回文对基因组维持和疾病的影响，这些重复序列如何导致染色体断裂， 在真核细胞中的重排在很大程度上是未知的。本研究的总体目标是阐明 酵母（Saccharomycescerevisiae）中回文序列的染色体断裂机制。的 该建议的中心假设是，回文序列的染色体脆性是由多重 核酸酶在细胞周期的不同阶段，并取决于形成的二级结构的类型。我们 初步数据表明，有三个不同的途径，其中二级结构可以启动双， 链断裂（DSB）形成和促进染色体不稳定性。一种途径涉及 Mre 11/Rad 50/Xrs 2（MRX）复合物和Sae 2，一种依赖于结构特异性Mus 81/Mms 4核酸酶， 另一种涉及未知的核酸酶在目标1中，我们将确定DSB形成所需的因素， MRX复合物和Sae 2。我们发现，完全回文结构是DSB的位点，而非准回文结构 并将测试核酸酶攻击发生在由MRX/Sae 2形成的发夹结构上的假设， S期完全回文。目标2将确定回文序列的参数， Mus 81/Mms 4核酸酶攻击二级结构的特异性。我们已经发现， 由活跃转录基因组成的完美回文部分依赖于Mus 81/Mms 4核酸酶。 这种核酸酶不会在另外两个非转录的回文区产生断裂。我们将决定 在Mus 81/Mms 4攻击中的转录，并将定义这种靶向所需的回文酶的参数。目的 3将鉴定负责MRX/Sae 2-和Mus 81/Mms 4-非依赖性的回文酶断裂的蛋白质。 关于回文脆性存在另一条由Lsm 2 -8复合体控制的途径的假设 并检测Cdc 28激酶。我们假设这一通路涉及一个未知的十字形解离酶 在细胞周期的G2期起作用。这项研究是创新的，因为它利用了 一套独特的敏感工具，可以识别所有导致回文脆性的核酸酶 并将决定它们的切割特异性。这一建议意义重大，因为它将阐明不良- 确定了产生染色体畸变的机制，这些染色体畸变是人类疾病的基础。

Palindromic sequences that adopt hairpin and cruciform structures are a potent source of chromosomal breakage and rearrangements, and play a significant role in the pathogenesis of diseases. In humans, for example, palindromes have been found at chromosomal breakpoints of non-recurrent and recurrent translocations that can cause Emanuel syndrome. In addition, palindrome-mediated chromosomal rearrangements cause several types of εγδβ thalassemia, X-linked congenital hypertrichosis syndrome and hereditary renal cell carcinoma. Finally, palindromes are implicated in the amplification of genes that promote tumorigenesis in colon and breast cancer, medulloblastoma and lymphoma. Despite the critical impact of palindromes on genome maintenance and disease, how these repeats cause chromosome breakage and rearrangements in eukaryotic cells is largely unknown. The overall objective of this research is to elucidate the mechanisms of chromosomal breakage at palindromic sequences in yeast, Saccharomyces cerevisiae. The central hypothesis of the proposal is that chromosomal fragility at palindromic sequences is caused by multiple nucleases at different stages of the cell cycle and is dependent on the type of secondary structure formed. Our preliminary data indicate that there are three distinct pathways by which secondary structures can initiate double- strand break (DSB) formation and promote chromosomal instability. One pathway involves the Mre11/Rad50/Xrs2 (MRX) complex and Sae2, one depends on the structure-specific Mus81/Mms4 nuclease, and one involves an unknown nuclease. In Aim 1, we will determine factors required for DSB formation by the MRX complex and Sae2. We have found that perfect palindromes but not quasi-palindromes are sites for DSB formation by MRX/Sae2 and will test the hypothesis that nuclease attack occurs on hairpin structures formed by perfect palindromes during S-phase. Aim 2 will identify parameters of palindromic sequences that determine the specificity of secondary-structure attack by the Mus81/Mms4 nuclease. We have found that breakage at a perfect palindrome composed of actively transcribed genes is partially dependent on the Mus81/Mms4 nuclease. This nuclease does not make breaks at two other nontranscribed palindromes. We will determine the role of transcription in Mus81/Mms4 attack and will define parameters of palindromes required for such targeting. Aim 3 will identify the protein(s) responsible for MRX/Sae2- and Mus81/Mms4-independent breakage of palindromes. The hypothesis that there is another pathway for palindrome fragility that is controlled by the Lsm2-8 complex and the Cdc28 kinase will be tested. We hypothesize that this pathway involves an unknown cruciform resolvase that operates during the G2 stage of the cell cycle. The proposed research is innovative because it utilizes a unique set of sensitive tools that will allow the identification of all nucleases contributing to palindrome fragility and will determine their cleavage specificity. This proposal is significant because it will elucidate the poorly- defined mechanisms that generate chromosomal aberrations that underlie human diseases.