ALTERNATIVE DNA DAMAGE CHECKPOINT PATHWAYS IN EUKARYOTES

研究了E.大肠杆菌对人类的感染 通过两个主要特征，细胞周期进程的阻滞和诱导 DNA修复所需的基因。 在真核生物中， (most尤其是ESR 1/MEC 1、RAD 3、MEI 41和ATM激酶）是保守的 并且是DNA损伤后细胞周期停滞所必需的。 ATM突变 或其他检查点基因，包括p53，导致增加 易患恶性肿瘤和对辐射反应的改变 疗法 一个新的人cDNA，CHES 1（检查点抑制因子1），编码一个成员 的叉头/翼障碍家庭，可以重建一个MEC 1- 当在酵母中表达时， 与世隔绝 CHES 1活性的分析与 激活一种新的替代检查点途径。 这项建议 基于这一观察，我们确定了这一过程所需的基因。 酵母中的替代检查点途径，并确定 哺乳动物细胞中的内源性和外源性CHES 1。 的 实验目的包括系统的遗传筛选， S.失去了替代途径的酿酒酵母。 在 平行，直接分离酵母和人类基因， 与CHES 1的结合将确定替代途径中的哪种基因产物是 被CHES 1激活。 这些基因的哺乳动物同源物 在这些目标中确定的特征将是他们的能力， 调节哺乳动物细胞对DNA损伤的反应。 鉴于 CHES 1抑制酵母DNA损伤敏感性的能力 对于MEC 1直接测定CHES 1是否外源表达 将抑制共济失调患者细胞中的检查点缺陷， 毛细血管扩张（AT）。 这将通过 用腺病毒构建体大量感染培养物， CHES 1. 这些后面的结果将证明是否激活一个 替代检查点途径可能用于治疗 AT患者或改变肿瘤对辐射的抵抗力， 其他DNA损伤剂

The response to DNA damage of organisms from E. coli to humans is marked by two major features, arrest of cell cycle progression, and induction of genes required for DNA repair. In eukaryotes, several gene products (most notably the ESR1/MEC1, RAD3, MEI41, and ATM kinases) are conserved and required for cell cycle arrest after DNA damage. Mutations in ATM or other checkpoint genes including p53 result in increased predisposition to malignancy and alteration in the response to radiation therapy. A novel human cDNA, CHES1 (Checkpoint Suppressor 1) encoding a member of the fork head/Winged Helix family which can reconstitute a MEC1- independent checkpoint when expressed in yeast has been recently isolated. Analysis of the activity of CHES1 is consistent with activation of a novel alternative checkpoint pathway. This proposal builds on this observation to define the genes required for this alternative checkpoint pathway in yeast and determine the activity of both endogenous and exogenous CHES1 in mammalian cells. The experimental aims include a systematic genetic screen to identify mutants of S. cerevisiae which have lost the alternative pathway. In parallel, direct isolation of both yeast and human genes which interact with CHES1 will define which gene product in the alternative pathway is being activated by CHES1. The mammalian homologs of the genes identified in these aims will be characterized for their ability to modulate the response to DNA damage of mammalian cells. Given the ability of CHES1 to suppress the DNA damage sensitivity of yeast deleted for MEC1 direct determination of whether exogenous expression of CHES1 will suppress the checkpoint defect in cells from patients with ataxia telangiectasia (AT) will be performed. This will be accomplished by mass infection of cultures with an adenoviral construct expressing CHES1. These latter results will demonstrate whether activation of an alternative checkpoint pathway might be used therapeutically for patients with AT or to alter the resistance of tumors to radiation and other DNA-damaging agents.