Soldatenkov Viatcheslav A, Chasovskikh Sergey, Potaman Vladimir N, Trofimova Irina, Smulson Mark E, Dritschilo Anatoly
Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007, USA.
J Biol Chem. 2002 Jan 4;277(1):665-70. doi: 10.1074/jbc.M108551200. Epub 2001 Oct 29.
Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme that plays roles in response to DNA damage, apoptosis, and genetic stability. Recent evidence has implicated PARP in transcription of eukaryotic genes. However, the existing paradigm tying PARP function to the presence of DNA strand breaks does not provide a mechanism by which it may be recruited to gene-regulating domains in the absence of DNA damage. Here we report that PARP can bind to the DNA secondary structures (hairpins) in heteroduplex DNA in a DNA end-independent fashion and that automodification of PARP in the presence of NAD+ inhibited its hairpin binding activity. Atomic force microscopic images show that in vitro PARP protein has a preference for the promoter region of the PARP gene in superhelical DNA where the dyad symmetry elements likely form hairpins according to DNase probing. Using a chromatin cross-linking and immunoprecipitation assay we show that PARP protein binds to the chromosomal PARP promoter in vivo. Reporter gene assays have revealed that the transcriptional activity of the PARP promoter is 4-5-fold greater in PARP knockout cells than in wild type fibroblasts. Reintroduction of vectors expressing full-length PARP protein or its truncated mutant (DNA-binding domain retained but lacking catalytic activity) into PARP(-/-) cells has conferred transcriptional down-regulation of the PARP gene promoter. These data provide support for PARP protein as a potent regulator of transcription including down-regulation of its own promoter.
聚（ADP - 核糖）聚合酶（PARP）是一种DNA结合酶，在DNA损伤应答、细胞凋亡和遗传稳定性方面发挥作用。最近的证据表明PARP参与真核基因的转录。然而，现有的将PARP功能与DNA链断裂联系起来的范例并未提供一种在无DNA损伤时将其招募到基因调控区域的机制。在此我们报告，PARP能以不依赖DNA末端的方式结合异源双链DNA中的DNA二级结构（发夹结构），并且在NAD⁺存在下PARP的自身修饰会抑制其发夹结合活性。原子力显微镜图像显示，体外PARP蛋白偏好超螺旋DNA中PARP基因的启动子区域，根据DNA酶探测，该区域的二元对称元件可能形成发夹结构。通过染色质交联和免疫沉淀试验，我们证明PARP蛋白在体内与染色体PARP启动子结合。报告基因检测显示，PARP启动子的转录活性在PARP基因敲除细胞中比在野生型成纤维细胞中高4 - 5倍。将表达全长PARP蛋白或其截短突变体（保留DNA结合结构域但缺乏催化活性）的载体重新导入PARP（- / -）细胞，可导致PARP基因启动子的转录下调。这些数据支持PARP蛋白作为转录的有效调节因子，包括对其自身启动子的下调作用。
