Ball K L, Lane D P
Department of Biochemistry, University of Dundee, UK.
Eur J Biochem. 1996 May 1;237(3):854-61. doi: 10.1111/j.1432-1033.1996.0854p.x.
The mechanism(s) whereby higher plants respond to environmental agents that damage their DNA, which leads to the arrest of cell division, is poorly understood. In mammalian cells, the tumour-suppressor protein p53 plays a central role in a DNA-damage-induced cell-cycle-checkpoint pathway by induction of transcription of a set of gene products that have a direct role in a DNA-damage-induced cell-cycle growth arrest. One such protein, p21WAF1, has been shown to be essential for radiation-induced growth arrest. There appear to be at least two cellular targets of p21WAF1 during checkpoint control, the G1-cyclin-dependent kinases (CDK) and proliferating-cell nuclear antigen (PCNA). The aim of the research reported here was to determine whether the interactions between the human growth inhibitor p21WAF1 and PCNA from plants and humans are conserved. If so, this would suggest that modulation of PCNA activity may play an important role in plant responses to DNA damage and would imply that functional homologue(s) of p21WAF1 exist in plants. We show that the p21WAF1-interaction domain of PCNA is conserved between humans and plants. A peptide that contains the site of human p21WAF1 that binds human PCNA has been used to precipitate PCNA from crude pea (Pisum savitum) extracts. We used the p21WAF1 peptide as an affinity matrix and showed that pea PCNA bound in a specific high-affinity manner. This finding was used to develop a purification protocol that allowed PCNA from plant tissue to be purified to homogeneity. Pure pea PCNA forms a stable complex with full-length human p21WAF1 and the specific amino acids of p21WAF1 required for the interaction have been identified. The critical residues were identical to those required for binding to human PCNA, which indicates that the interaction of human p21WAF1 with PCNA is highly conserved at each amino acid position between pea and human.
高等植物对破坏其DNA并导致细胞分裂停滞的环境因子作出反应的机制，目前了解甚少。在哺乳动物细胞中，肿瘤抑制蛋白p53在DNA损伤诱导的细胞周期检查点途径中起核心作用，它通过诱导一组基因产物的转录来实现这一作用，这些基因产物在DNA损伤诱导的细胞周期生长停滞中具有直接作用。一种这样的蛋白质，p21WAF1，已被证明对辐射诱导的生长停滞至关重要。在检查点控制过程中，p21WAF1似乎至少有两个细胞靶点，即G1期细胞周期蛋白依赖性激酶（CDK）和增殖细胞核抗原（PCNA）。本文报道的研究目的是确定人类生长抑制剂p21WAF1与植物和人类的PCNA之间的相互作用是否保守。如果是这样，这将表明PCNA活性的调节可能在植物对DNA损伤的反应中起重要作用，并且意味着p21WAF1在植物中存在功能同源物。我们表明，PCNA的p21WAF1相互作用结构域在人类和植物之间是保守的。一个包含人类p21WAF1与人类PCNA结合位点的肽已被用于从豌豆（Pisum savitum）粗提物中沉淀PCNA。我们使用p21WAF1肽作为亲和基质，结果表明豌豆PCNA以特定的高亲和力方式结合。这一发现被用于开发一种纯化方案，该方案可将植物组织中的PCNA纯化至同质。纯豌豆PCNA与全长人类p21WAF1形成稳定复合物，并且已经确定了相互作用所需的p21WAF1的特定氨基酸。关键残基与结合人类PCNA所需的残基相同，这表明豌豆和人类之间，人类p21WAF1与PCNA的相互作用在每个氨基酸位置上都高度保守。
