Chen I T, Smith M L, O'Connor P M, Fornace A J
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Oncogene. 1995 Nov 16;11(10):1931-7.
We have previously shown (Smith et al., 1994) that antibodies raised against the growth arrest and DNA damage inducible protein Gadd45 co-precipitate proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair. Here we demonstrate that Gadd45 can directly bind to PCNA using a Far-western blotting approach. In this assay, a Gadd45 bacterial expression vector was modified to allow synthesis of purified 32P-labeled Gadd45 fusion protein. This protein was used to detect filter bound PCNA protein, while filter bound Gadd45 protein could also be detected by free PCNA molecules. Using recombinant proteins in conjunction with immunoprecipitation and immunoblotting, we show that Gadd45 competes with p21 for binding to PCNA and conversely, p21 blocks the ability of Gadd45 to bind PCNA. In addition, p21 appears to disrupt PCNA trimers whereas Gadd45 has a lesser effect. PCNA trimer disruption was also observed in UV-irradiated cells but not in repair-defective xeroderma pigmentosum group A (XP-A) cells.
我们之前已经证明（史密斯等人，1994年），针对生长停滞和DNA损伤诱导蛋白Gadd45产生的抗体能与增殖细胞核抗原（PCNA）共沉淀，PCNA是一种参与DNA复制和修复的蛋白质。在此我们证明，使用Far-western印迹法，Gadd45可直接与PCNA结合。在该实验中，对Gadd45细菌表达载体进行了改造，以使其能合成纯化的32P标记的Gadd45融合蛋白。该蛋白用于检测滤膜结合的PCNA蛋白，而游离的PCNA分子也能检测到滤膜结合的Gadd45蛋白。通过结合使用重组蛋白以及免疫沉淀和免疫印迹技术，我们发现Gadd45与p21竞争结合PCNA，反之，p21会阻断Gadd45结合PCNA的能力。此外，p21似乎会破坏PCNA三聚体，而Gadd45的影响较小。在紫外线照射的细胞中也观察到了PCNA三聚体的破坏，但在修复缺陷型A型着色性干皮病（XP-A）细胞中未观察到。
