Okazaki fragment maturation: mutagenesis and cell survival

冈崎片段成熟：诱变和细胞存活

基本信息

批准号：
10636417
负责人：
BINGHUI SHEN
金额：
$ 54.78万
依托单位：
BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2028-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10636417
关键词：
3&apos -nuclease Applications Grants CHEK1 gene CHEK2 gene Cancer Model Cell Death Cell Maturation Cell Proliferation Cell Senescence Induction Cell Survival Cells DNA Damage DNA Sequence Alteration DNA biosynthesis DNA damage checkpoint Data Drug resistance Enzymes Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Evolution Genetic Screening Genomic Segment Goals Growth Human Impairment In Vitro Malignant Epithelial Cell Mammalian Cell Measures Modeling Molecular Mus Mutagenesis Mutate Mutation Non-Small-Cell Lung Carcinoma Okazaki fragments Pathway interactions Phosphorylation Probability Process RTH-1 Nuclease Radiation therapy Resistance Role Signal Induction Signal Transduction Site Stress Structure Supporting Cell Temperature Testing Yeasts acquired drug resistance anti-cancer cancer cell cancer therapy chemotherapy genome-wide helicase in vitro activity lung cancer cell mouse model mutant nuclease prevent recruit replication stress response therapy development therapy resistant yeast genetics

项目摘要

ABSTRACT The long-term goal of this project is to define the molecular mechanisms of an error-prone, stress-induced Okazaki fragment maturation (OFM) pathway by which cancer cells counteract replication stress and survive. Replication stress is a hallmark of cancer cells and has been considered the Achilles' heel for cancer treatment such as radio- and chemotherapy. Under elevated temperature stress, yeast cells mutant for flap endonuclease 1 (FEN1 in humans or RAD27 in yeast) activate DNA damage response pathways to block cell proliferation and induce cell senescence and death; however, a subpopulation of cells can overcome these barriers and escape otherwise lethal conditions. Genome-wide mutations and rearrangements have been suggested as a major molecular mechanism that drives this evolution. However, how such spontaneous mutations are acquired in cells under replication stress is a long-standing question. Recently, we identified an error-prone, 3' flap OFM pathway that is activated in response to stress to support cell survival and fuel cellular evolution; its induction leads to genome-wide mutagenesis and suppression of restrictive growth temperature-induced lethality, a process mimicking that of cancer cells acquiring drug resistance. This led us to a model in which OFM can go in two ways, which may dictate the fate of cells, including human cancer cells: a 5' flap-based, error-free process or an alternative 3' flap-based, stress-induced, and error-prone process. However, key components that drive such flap dynamics remain undefined. The objectives of the proposed project are to define the key enzymes that catalyze 3' flap formation and cleavage in mammalian cells and to provide proof of concept that suppressing alternative 3' flap OFM can prevent drug resistance in human cancer cells. Further preliminary data gathered to support this grant application show that 3' flap OFM is conserved in both yeast and human cells. We observed that anti-cancer EGFR tyrosine kinase inhibitors activated the ATM/CHK2 DNA damage checkpoints in human lung cancer cells. Using yeast genetic screening, we identified Pif1 (PIF1 in humans) and Sgs1 (BLM and WRN in humans) as helicases for 5' to 3' flap transformation and Rad1 (XPF in humans) and Mus81 (MUS81 in humans) as 3' nucleases for 3' flap cleavage, in addition to the 3' nuclease activity of Pol . Therefore, our central hypothesis is that unprocessed 5' flaps in mammalian cells activate ATM/ATR and CHK1/2 signaling to recruit and stimulate PIF1, BLM, and WRN and/or other helicases for transforming 5' flaps into 3' flaps for nucleolytic degradation by 3' nucleases including Pol , XPF, and/or MUS81, and that blocking the 3' flap OFM pathway will suppress DNA mutations and thus prevent drug resistance. To test this, we will: i) determine the roles of helicases PIF1, BLM, and WRN in 3' flap formation and induction of alternative OFM; ii) define the functional distribution of 3' nucleases Pol , XPF, and MUS81 in processing 3' flaps with or without secondary structures during 3' flap OFM; and iii) define the extent to which stress-activated ATM/CHK2 signaling induces 3' flap OFM and mutations to support cancer cell survival and promote drug resistance.

抽象的该项目的长期目标是定义容易出错、压力诱导的分子机制冈崎片段成熟（OFM）途径，癌细胞通过该途径抵消复制压力并存活。复制压力是癌细胞的标志，被认为是癌症治疗的致命弱点例如，在高温应激下，酵母细胞会发生瓣状核酸内切酶 1 突变。（人类中的 FEN1 或酵母中的 RAD27）激活 DNA 损伤反应途径以阻止细胞增殖和诱导细胞衰老和死亡；然而，细胞亚群可以克服这些障碍并逃脱其他致命的情况已被认为是主要的基因组突变和重排。然而，这种自发突变是如何在细胞中获得的。最近，我们发现了一个容易出错的 3' 瓣 OFM 通路。它因应激而被激活，以支持细胞生存并促进细胞进化；全基因组诱变和限制性生长温度诱导致死的抑制，一个过程模仿癌细胞获得耐药性的过程，这使我们建立了一个 OFM 可以分为两部分的模型。方式，这可能决定细胞的命运，包括人类癌细胞：基于 5' 瓣的无差错过程或替代的基于 3' 瓣的、应力引起的且容易出错的过程然而，驱动这种过程的关键组件。皮瓣动力学仍未定义。拟议项目的目标是定义关键酶。催化哺乳动物细胞中 3' 瓣的形成和裂解，并提供抑制替代 3' 瓣 OFM 可以预防人类癌细胞的耐药性。支持这项资助申请表明我们观察到 3' 瓣 OFM 在酵母和人类细胞中都是保守的。抗癌 EGFR 酪氨酸激酶抑制剂激活人类 ATM/CHK2 DNA 损伤检查点通过酵母基因筛选，我们鉴定出了 Pif1（人类中的 PIF1）和 Sgs1（BLM 和 WRN）。人类中）作为 5' 至 3' 瓣转化的解旋酶以及 Rad1（人类中的 XPF）和 Mus81（人类中的 MUS81）人类）作为 3' 瓣切割的 3' 核酸酶，除了 Pol  的 3' 核酸酶活性之外，因此，我们的中心。假设哺乳动物细胞中未经加工的 5' 瓣激活 ATM/ATR 和 CHK1/2 信号传导以招募并刺激 PIF1、BLM 和 WRN 和/或其他解旋酶，将 5' 皮瓣转化为 3' 皮瓣以进行溶核被 3' 核酸酶（包括 Pol 、XPF 和/或 MUS81）降解，并阻断 3' 瓣 OFM 通路会抑制 DNA 突变，从而防止耐药性。为了测试这一点，我们将： i) 确定的作用。解旋酶 PIF1、BLM 和 WRN 在 3' 瓣形成和替代 OFM 诱导中的作用 ii) 定义功能； 3' 核酸酶 Pol 、XPF 和 MUS81 在处理具有或不具有二级结构的 3' 瓣中的分布在 3' 瓣 OFM 期间；以及 iii) 定义应激激活的 ATM/CHK2 信号传导诱导 3' 瓣 OFM 的程度以及支持癌细胞存活和促进耐药性的突变。