DYNAMICS AND FUNCTION OF G4 DNA

描述(申请人提供)：富含G的DNA可以形成由鸟嘌呤之间的相互作用稳定的结构，称为G4DNA。这些结构在体外已经得到了广泛的研究，但直到最近才被证明在体内形成。我的实验室最近直接鉴定出在体内形成的G4DNA。在这一结果的基础上，我们现在建议详细研究G4DNA是如何形成的，它是如何从活细胞中消除的，以及G4DNA是否有助于基因组的不稳定。为此，我们将追求以下具体目标：(1)我们将研究G4DNA是否形成于两个富含G的基因，c-myc和rDNA重复序列；(2)我们将研究G4DNA形成是否有助于酵母基因组的不稳定性；(3)我们将研究人类细胞中富含G重复序列的不稳定性，以及BLM解旋酶在其维持中的功能；(4)我们将鉴定RECQ家族解旋酶中指定G4DNA相互作用的保守基序(S)，并创建相应的突变体来研究该家族酶在体内的功能；，以及(5)我们将询问错配修复因子如何参与G4DNA的消除。这项提议中的实验结果将扩大我们对活细胞中G4DNA形成的理解，表征促进其消除的途径，并确定其对导致遗传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.