Okazaki fragment maturation: mutagenesis and cell survival

冈崎片段成熟：诱变和细胞存活

• 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
• 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' -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

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的，应力诱导的分子机制 Okazaki碎片成熟（OFM）途径，癌细胞抵消复制应力并存活。 复制应力是癌细胞的标志，被认为是致命癌治疗的阿喀琉斯脚跟 例如放射和化学疗法。在升高的温度应力下，酵母细胞突变的flap核酸内切酶1 （人类中的Fen1或酵母中的Rad27）激活DNA损伤响应途径，以阻止细胞增殖和 诱导细胞感应和死亡；但是，细胞的亚群可以克服这些障碍并逃脱 否则致命的条件。全基因组突变和重排被认为是主要的 驱动这种进化的分子机制。但是，如何在细胞中获取这种赞助突变 在复制压力下是一个长期的问题。最近，我们确定了一个容易出错的3'襟翼。 这是为了响应压力来支持细胞存活和燃料细胞进化而激活的；它的归纳导致 全基因组诱变和抑制限制性生长温度引起的致死性，这是一个过程 模仿癌细胞加速耐药性。这导致我们达到了一个模型，其中Ofm可以分为两个 可能决定细胞命运的方法，包括人类癌细胞：基于5'瓣的，无错误的过程或 替代3'襟翼基于应力诱导的和容易出错的过程。但是，驱动这样的关键组件 皮瓣动态仍然不确定。拟议项目的目标是定义关键酶 催化哺乳动物细胞中的3'皮瓣形成和裂解，并提供抑制概念证明 替代3'襟翼可以防止人类癌细胞中的耐药性。进一步收集到 支持此赠款应用显示，在酵母和人类细胞中均保存3'襟翼。我们观察到 抗癌EGFR酪氨酸激酶抑制剂激活了人类的ATM/CHK2 DNA损伤检查点 肺癌细胞。使用酵母基因筛选，我们鉴定了PIF1（人类中的PIF1）和SGS1（BLM和WRN 在人类中）作为5'至3'瓣转换的解旋酶，Rad1（人类XPF）和MUS81（MUS81中的MUS81 除3'的核酸酶活性外，人类）作为3'瓣裂解的3'核。因此，我们的中心 假设是哺乳动物细胞中未加工的5'襟翼激活ATM/ATR和CHK1/2信号以募集 并刺激PIF1，BLM和WRN和/或其他解旋酶，用于将5'襟翼转化为3'襟翼以进行核酸化 3'核的降解，包括pol，XPF和/或MUS81，并阻止了3'襟翼的途径 将抑制DNA突变，从而防止耐药性。要测试这一点，我们将：i）确定 在3'皮瓣形成中的旋转酶PIF1，BLM和WRN以及替代ofm的诱导； ii）定义功能 在处理有或没有次级结构的3'襟翼中，在处理3'的襟翼中的3'核电pol，XPF和MUS81的分布 在3'襟翼中； iii）定义应力激活的ATM/CHK2信号传导的程度3'襟翼 和支持癌细胞存活并促进耐药性的突变。