ALTERNATIVE DNA DAMAGE CHECKPOINT PATHWAYS IN EUKARYOTES

从大肠杆菌到人类的生物对DNA损伤的反应是显著的 通过两个主要特征，阻止细胞周期进程和诱导 DNA修复所需的基因。在真核生物中，有几种基因产物 (最值得注意的是ESR1/Mec1、Rad3、MEI41和ATM激酶)是保守的 DNA损伤后细胞周期停滞所必需的。自动取款机中的突变 或包括P53在内的其他检查点基因会导致 恶性肿瘤易感性与辐射反应的改变 心理治疗。 编码一个新成员的人类新基因CHES1(Checkpoint Suppressor 1) 叉头/有翼螺旋家族可以重建Mec1- 在酵母中表达的独立检查点最近已经被 与世隔绝。对CHES1活性的分析与 激活一条新的替代检查点途径。这项建议 建立在这一观察的基础上，以定义这一过程所需的基因 在酵母中选择检查点途径并测定其活性 哺乳动物细胞中的内源性和外源性CHES1。这个 实验目标包括系统的基因筛查，以识别 失去替代途径的酿酒酵母突变体。在……里面 平行、直接分离酵母和人类相互作用的基因 CHES1将定义替代途径中的哪种基因产物 被CHES1激活。这些基因的哺乳动物同源基因 在这些目标中确定的将以他们的能力为特征 调节哺乳动物细胞对DNA损伤的反应。给定 CHES1对缺失酵母DNA损伤敏感性的抑制作用 For Mec1直接测定CHES1是否外源表达 将抑制共济失调患者细胞中的检查点缺陷 将进行毛细血管扩张术(AT)。这将通过以下方式实现 重组腺病毒载体在培养上的大规模感染 CHES1。这些后一种结果将证明激活一个 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 其他破坏DNA的物质。

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.