Okazaki fragment maturation: mutagenesis and cell survival

摘要 这个项目的长期目标是定义一种容易出错的、应激诱导的 冈崎片段成熟(OFM)途径，癌细胞通过该途径中和复制压力和生存。 复制压力是癌细胞的一个标志，被认为是癌症治疗的致命弱点 比如放射治疗和化疗。高温胁迫下酵母细胞翻盖内切酶1的突变 (人类的FEN1或酵母的RAD27)激活DNA损伤反应通路，阻止细胞增殖和 导致细胞衰老和死亡；然而，一群细胞可以克服这些障碍并逃脱 否则是致命的条件。全基因组的突变和重排被认为是主要的 驱动这一进化的分子机制。然而，这种自发突变是如何在细胞中获得的 复制压力是一个长期存在的问题。最近，我们发现了一种容易出错的3‘瓣的FM通路 在应激反应中被激活，以支持细胞存活和推动细胞进化；它的诱导导致 全基因组突变和抑制限制性生长温度诱导的致死，这是一个过程 模仿癌细胞获得抗药性的过程。这让我们找到了一种模型，在这种模型中，OFM可以分成两部分 方式，这可能决定细胞的命运，包括人类癌细胞：5‘瓣为基础的，无错误的过程或 另一种基于3‘瓣、应力诱导和容易出错的过程。然而，推动这一趋势的关键组件 襟翼动力学仍未定义。拟议项目的目标是定义关键的酶 在哺乳动物细胞中催化3‘瓣的形成和分裂，并提供抑制 替代的3‘瓣可以防止人类癌细胞产生耐药性。进一步收集初步数据以 支持这项资助申请表明，3‘瓣OFM在酵母和人类细胞中都是保守的。我们观察到 抗癌EGFR酪氨酸激酶抑制剂激活人ATM/Chk2 DNA损伤检查点 肺癌细胞。通过酵母遗传筛选，我们鉴定了Pif1(人类中的PIF1)和SGS1(BLm和WRN 在人类中)作为5‘到3’瓣转化的解旋酶以及Rad1(人中的XPF)和MUS81(MUS81中的 除了POL的3‘核酸酶活性外，Pol DNA还具有3’端核酸酶活性。因此，我们的中央 假说认为，哺乳动物细胞中未经处理的5‘端瓣激活ATM/ATR和CHK1/2信号以招募 并刺激PIF1、BLm、WRN和/或其他螺旋酶将5‘瓣转化为3’瓣进行核溶解 包括Pol、Xpf和/或MUS81在内的3‘核酸酶的降解，以及阻断FM途径的3’端折叠 将抑制DNA突变，从而防止耐药性。为了测试这一点，我们将：i)确定 解旋酶PIF1、BLm和WRN在3‘瓣的形成和替代OFM的诱导中；ii)定义 3‘核酸酶Pol、XpF和MUS81在有无二级结构的3’皮瓣中的分布 以及iii)定义应激激活的ATM/Chk2信号诱导3‘翻转的程度 和突变，以支持癌细胞存活和促进耐药性。

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.