ROLE OF DNA STRAND BREAKS AND PARP ON IONIZING RADIATION INDUCED APOPTOSIS

描述(由申请人提供)：我们建议继续从分子上阐明PARP在辐射(IR)诱导的细胞凋亡中的具体作用。它基于一个总体假设，即蛋白质的共价p(ADP-核糖)n通过长的、带负电的聚合物链使目标DNA结合蛋白“DNA恐惧症”，并阻止它们与DNA结合。具体目的1研究细胞凋亡过程中人类细胞内钙/镁结合蛋白DNASIL3的调控，这是导致基因组DNA晚期核小体降解的原因。当DNASIL3处于p(ADP-核糖)n状态时，DNASIL3被抑制，因此在体外被调节。在成功克隆了人DNASIL3基因后，我们希望通过PARP调节在IR诱导的细胞死亡中阐明其在体内的作用。导入抗caspase的PARP的细胞将被用来评估IR诱导的DNASIL3共价结合的稳定的、未切割的聚合物的后果。针对PARP糖水解酶(PARG)的可诱导反义和正义cDNA2的方法将建立稳定的细胞系。这种酶专门从DNAS1L3和P53等蛋白质中切割PAR。因此，PARG要么被消除，要么在细胞中过度表达。在最初的案例中，我们将测试缺乏从DNAS1L3和P53中切割PAR的能力将如何影响p(ADP-R)n对核蛋白的调节。将进行免费的PARG过表达实验，以测试稳定的p(ADP-R)n状态 DNASIL3和P53。具体目标3将集中在DFF45，一种核酸酶抑制剂DFF40。该核酸酶在细胞凋亡早期50kb DNA裂解的染色质重组中起着关键作用。来自DFF45-/-和+/+小鼠的细胞将被用来建立DFF45-/-细胞对链断裂诱导的细胞死亡的保护机制。这涉及到关键的凋亡事件和PARP-1相关的扩增环。我们将测试IR是否导致辐射诱导的细胞凋亡过程中PARP-1的双相激活，在直接DNA损伤后立即，然后在Dff激活和50kb DNA断裂之后。

DESCRIPTION (provided by applicant): We propose to continue to molecularly clarify specific roles of PARP in radiation (IR)-induced apoptosis. It is based on the overall hypothesis that the covalent p(ADP-ribose)n of proteins by long, negatively charged chains of polymer renders target DNA-binding proteins "DNA phobic" and prevents their binding to DNA. Specific Aim 1 focuses on the regulation of human Ca2+/Mg2+ DNASIL3 during apoptosis, which causes late nucleosomal degradation of genomic DNA. DNASIL3 is inhibited and hence regulated in vitro when in the p(ADP-ribose)n state. Having successfully cloned the human DNASIL3, we wish to clarify its role in vivo via PARP regulation in IR-induced cell death. Cells transfected with a caspase-resistant PARP will be used to assess the consequences during IR-induced apoptosis of a now stable, uncleaved polymer, covalently bound to DNASIL3. Approaches in Specific Aim 2 will establish cell lines, stably transfected with inducible antisense and sense cDNA for PARP glycohydrolase (PARG). This enzyme specifically cleaves PAR from proteins such as DNAS1L3 and p53. Accordingly, PARG will be either eliminated or over expressed in cells. In the initial case we will test how the lack of ability to cleave PAR from DNAS1L3 and p53 will influence the regulation imposed by p(ADP-R)n on nuclear proteins. Complimentary PARG overexpression experiments will be performed to test the effects of a stable p(ADP-R)n state of the DNASIL3 and p53. Specific Aim 3 will focus on DFF45, an inhibitor of the nuclease, DFF40. This nuclease plays a key role in chromatin restructuring by 50 Kb DNA cleavage early in apoptosis. Cells derived from DFF45-/- and +/+ mice will be used to establish the mechanism underlying the protection of DFF45-/- cells against strand breaks-induced cell death. This involves key apoptotic events and a PARP-1-associated amplification loop. We will test whether IR causes a biphasic activation of PARP-1 during radiation-induced apoptosis immediately after direct DNA damage and secondly after DFF activation and the generation of 50 kb DNA breaks.