Okazaki fragment maturation: mutagenesis and cell survival

摘要 该项目的长期目标是确定一个容易出错的，压力诱导的 冈崎片段成熟（OFM）途径，癌细胞通过该途径抵消复制应激并存活。 复制压力是癌细胞的一个标志，一直被认为是癌症治疗的致命弱点 如放射治疗和化学治疗。高温胁迫下酵母细胞flap内切酶1突变 (FEN1在人类中或酵母中的RAD 27）激活DNA损伤反应途径以阻断细胞增殖， 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逸 否则会致命全基因组突变和重排被认为是一个主要的 驱动这种进化的分子机制。然而，这种自发突变是如何在细胞中获得的， 是一个长期存在的问题。最近，我们发现了一个易错的3'瓣OFM通路， 在应激反应中被激活，以支持细胞存活和促进细胞进化;其诱导导致 全基因组诱变和抑制限制性生长温度诱导的致死性， 模仿癌细胞获得抗药性的过程。这让我们想到了一个模型，在这个模型中， 方法，这可能决定细胞的命运，包括人类癌细胞：一个5'皮瓣为基础的，无差错的过程或一个 可选的3 ′基于襟翼的、应力诱导的和易于出错的过程。然而，推动这种增长的关键因素 襟翼动力学仍然不确定。该项目的目标是确定关键酶， 在哺乳动物细胞中催化3 ′瓣形成和切割，并提供抑制 替代3'瓣OFM可以防止人类癌细胞的耐药性。进一步收集初步数据， 支持该授权申请表明3 ′瓣OFM在酵母和人细胞中都是保守的。我们观察到 抗癌EGFR酪氨酸激酶抑制剂激活人类ATM/CHK 2 DNA损伤检查点 肺癌细胞使用酵母遗传筛选，我们鉴定了Pif 1（人类PIF 1）和Sgs 1（BLM和WRN 在人类中）作为解旋酶用于5'至3'瓣转化，以及Radl（在人类中的XPF）和Mus 81（在人类中的MUS 81）作为解旋酶用于5 '至3'瓣转化。 人）作为3'核酸酶用于3'瓣切割，除了Poll β的3'核酸酶活性之外。因此，我们的中央 假设哺乳动物细胞中未处理的5'瓣激活ATM/ATR和CHK 1/2信号以募集 并刺激PIF 1、BLM和WRN和/或其它解旋酶，用于将5 ′瓣转化为3 ′瓣，用于溶核 通过3'核酸酶（包括Pol β、XPF和/或MUS 81）降解，以及阻断3'瓣OFM途径 会抑制DNA突变从而防止耐药性为了验证这一点，我们将：i）确定 解旋酶PIF 1、BLM和WRN在3'瓣形成和替代OFM的诱导中的作用; ii）定义了功能性的 3 ′核酸酶Pol ′、XPF和MUS 81在处理有或无二级结构的3 ′皮瓣中的分布 iii）定义应激激活的ATM/CHK 2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。

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.