Translesion synthesis DNA polymerases and genome instability

摘要 这种竞争性更新应用试图研究跨损伤合成DNA聚合酶在 与减数分裂细胞分裂相关的突变。与只影响个体的体细胞突变不同， 在减数分裂过程中发生的DNA变化会传递给下一代，并导致 遗传性疾病。在过去的几十年里，随着生育年龄的稳步上升， 人类群体中的Novo生殖系突变一直在不断上升。由此产生的遗传性 疾病给个人、家庭和社会带来了沉重的负担，因为患者往往 在很小的时候就出现了多种医学问题，需要专门的终身护理。其作用机制 对生殖系突变的产生负有责任的人知之甚少，因为几乎所有的 突变使用有丝分裂细胞。在这笔赠款的前几个周期中，我们发现并探索了一部小说 容易出错的DNA聚合酶ζ(POLζ)被招募到DNA复制叉处的突变途径 在小发夹DNA结构处停滞，并促进这些结构的旁路，产生一种特征 突变特征。这一途径在健康的有丝分裂细胞中是沉默的，因为强劲的正常复制 小型次级结构不会对机械造成明显的阻碍。依赖于ζ的POL容易出错 然而，当内在或环境压力促进叉子失速时，结构旁路成为一个因素。 出乎意料的是，我们的初步数据表明，这一途径在正常减数分裂过程中也被激活，并且 癌症易感基因反复发生胚系突变的可能来源。我们将通过以下方式验证这一假设 追求三个具体目标。在目标1中，我们将定义POLζ依赖途径对 种系和减数分裂特异的突变。在目标2中，我们将使用与遗传性基因连锁的人类极点基因 以结直肠癌易感综合征为模型识别减数分裂特异性突变热点和 定义它们与POLζ依赖的发夹旁路的关系。在目标3中，我们将确定 环境DNA损伤剂和复制抑制剂对POL-ζ依赖的蓄积的影响 配子发生过程中的突变。酵母、小鼠和人类细胞模型将用于这些研究， 酵母菌系统为机械分析提供了最大的动力，鼠标提供了机会 实验研究哺乳动物体内减数分裂过程中的诱变，以及对人类样本的数据 建立与疾病的终极联系。我们期待着对这一机制有新的基础知识 影响后代的生殖系突变。我们还希望了解以下原因 某些致病生殖系变异的频繁从头形成及其环境影响 各种因素。

Abstract This competitive renewal application seeks to investigate the role of translesion synthesis DNA polymerases in mutagenesis associated with meiotic cell divisions. Unlike somatic mutations that only impact the individual, DNA changes occurring during meiosis are transmitted to the next generations and lead to the development of hereditary diseases. With the childbirth age steadily increasing over the past decades, the frequency of de novo germline mutations in the human population has continuously been on the rise. The resulting hereditary diseases present a significant burden for the individuals, families and the society, since the patients often develop multiple medical problems at an early age and require specialized life-long care. The mechanisms responsible for the generation of germline mutations are poorly understood, as nearly all mechanistic studies of mutagenesis employ mitotic cells. In the previous cycles of this grant, we discovered and explored a novel mutagenesis pathway wherein error-prone DNA polymerase ζ (Polζ) is recruited to DNA replication forks stalled at small hairpin DNA structures and facilitates the bypass of these structures, producing a characteristic mutational signature. This pathway is silent in healthy mitotic cells because the robust normal replication machinery is not significantly impeded by the small secondary structures. The Polζ-dependent error-prone structure bypass, however, becomes a factor when intrinsic or environmental stressors promote fork stalling. Unexpectedly, our preliminary data suggested that this pathway is also activated during normal meiosis and is a likely source of recurrent germline mutations in cancer predisposition genes. We will test this hypothesis by pursuing three Specific Aims. In Aim 1, we will define the contribution of the Polζ-dependent pathway to germline- and meiosis-specific mutagenesis. In Aim 2, we will use the human POLE gene linked to a hereditary colorectal cancer predisposition syndrome as a model to identify meiosis-specific hotspots of mutagenesis and define their relationship to Polζ-dependent hairpin bypass. In Aim 3, we will determine the effects of environmental DNA damaging agents and replication inhibitors on the accumulation of Polζ-dependent mutations during gametogenesis. Yeast, mouse and human cell models will be used in these studies, with the yeast system providing the most power for mechanistic analysis, the mouse providing the opportunity to experimentally study mutagenesis during mammalian meiosis in vivo, and the data on human samples establishing the ultimate link to disease. We expect to gain new fundamental knowledge on the mechanism of mutagenesis in the germline that impacts future generations. We also expect to understand the reasons for frequent de novo formation of some disease-causing germline variants and the effects of environmental factors.