DYNAMICS AND FUNCTION OF G4 DNA

描述（由申请人提供）：富含g的DNA可以通过鸟嘌呤相互作用形成稳定的结构，称为G4 DNA。这些结构已在体外广泛研究，但直到最近才显示在体内形成。我的实验室最近直接鉴定了体内形成的G4 DNA。在此结果的基础上，我们现在建议详细研究G4 DNA如何形成，如何从活细胞中消除，以及G4 DNA是否有助于基因组不稳定性。为此，我们将追求以下具体目标：(1)我们将询问G4 DNA是否在两个富含g的基因，c-myc和rDNA重复序列上形成；(2)我们将探讨G4 DNA的形成是否会导致酵母基因组的不稳定性；(3)研究人类细胞中富g重复序列的不稳定性，以及BLM解旋酶在维持其稳定性中的作用；(4)我们将在RecQ家族解旋酶中找到指定G4 DNA相互作用的保守基序，并创建相应的突变体来研究该家族酶的体内功能；(5)错配修复因子如何参与G4 DNA的消除。本提案中的实验结果将扩展我们对活细胞中G4 DNA形成的理解，表征促进其消除的途径，并确定其对导致遗传疾病和恶性肿瘤发展的基因组不稳定性的贡献。

DESCRIPTION (provided by applicant): G-rich DNA can form structures stabilized by interactions between guanines, referred to as G4 DNA. These structures have been widely studied in vitro, but until recently had not been shown to form in vivo. My laboratory has very recently directly identified G4 DNA formed in vivo. Building upon and extending this result, we now propose to study in detail how G4 DNA forms, how it is eliminated from living cells, and whether G4 DNA contributes to genomic instability. To that end we will pursue the following specific aims: (1) We will ask if G4 DNA forms at two G-rich genes, c-myc and the rDNA repeats; (2) we will ask whether G4 DNA formation contributes to genomic instability in yeast; (3) we will study instability of G-rich repeats in human cells, and the functions of BLM helicase in their maintenance; (4) we will identify the conserved motif(s) within RecQ family helicases that specify G4 DNA interaction, and create the corresponding mutants to study functions of enzymes in this family in vivo;, and (5) we will ask how mismatch repair factors participate in elimination of G4 DNA. Results from experiments in this proposal will extend our understanding of G4 DNA formation in living cells, characterize pathways that promote its elimination, and establish its contribution to genomic instability that leads to genetic disease and development of malignancy.