Fukuda K, Morioka H, Imajou S, Ikeda S, Ohtsuka E, Tsurimoto T
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
J Biol Chem. 1995 Sep 22;270(38):22527-34. doi: 10.1074/jbc.270.38.22527.
Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymerase delta (pol delta). Due to the functional and structural similarity with the beta-subunit of Escherichia coli DNA polymerase III, it has been proposed that PCNA would act as a molecular clamp during DNA synthesis. By site-directed mutagenesis and biochemical analyses, we have studied the functional domains of human PCNA required for stimulation of replication factor C (RF-C) ATPase and DNA synthesis by pol delta. Short deletions from either the N or C termini caused drastic changes in extraction and chromatographic behaviors, suggesting that both of these terminal regions are crucial to fold the tertiary structure of PCNA. The short C-terminal stretch from Lys254 to Glu256 is necessary for stimulation of RF-C ATPase activity, but not for stimulation of DNA synthesis by pol delta. Nine basic amino acids that are essential for activating DNA synthesis by pol delta are positioned at the internal alpha-helices of PCNA. This result is in good agreement with the observation that PCNA has a ring structure similar to the beta-subunit and clamps a template DNA through this positively charged internal surface. Several other charged amino acids are also required to stimulate either RF-C ATPase or pol delta DNA synthesis. Some of them are positioned at loops which are exposed on one of the side surface of PCNA adjacent to the C-terminal loop. In addition, the beta-sheets composing the intermolecular interface of the trimeric PCNA are important for interaction with pol delta. Therefore, the outer surface of PCNA has multiple functional surfaces which are responsible for the interaction with multiple factors. Furthermore, the two side surfaces seem to be functionally distinguishable, and this may determine the orientation of tracking PCNA along the DNA.
增殖细胞核抗原（PCNA）对于真核生物DNA复制至关重要，并且作为DNA聚合酶δ（pol δ）的持续合成因子发挥作用。由于其与大肠杆菌DNA聚合酶III的β亚基在功能和结构上具有相似性，有人提出PCNA在DNA合成过程中会充当分子夹子。通过定点诱变和生化分析，我们研究了人PCNA中刺激复制因子C（RF-C）ATP酶活性以及pol δ介导的DNA合成所必需的功能结构域。从N端或C端进行的短片段缺失导致提取和色谱行为发生剧烈变化，这表明这两个末端区域对于折叠PCNA的三级结构都至关重要。从赖氨酸254到谷氨酸256的短C末端片段对于刺激RF-C ATP酶活性是必需的，但对于pol δ介导的DNA合成刺激并非必需。激活pol δ介导的DNA合成所必需的九个碱性氨基酸位于PCNA的内部α螺旋上。这一结果与以下观察结果非常吻合：PCNA具有与β亚基相似的环状结构，并通过这个带正电荷的内表面夹住模板DNA。还需要其他几个带电荷的氨基酸来刺激RF-C ATP酶或pol δ介导的DNA合成。其中一些位于PCNA相邻于C末端环的一个侧面暴露的环上。此外，构成三聚体PCNA分子间界面的β折叠对于与pol δ的相互作用很重要。因此，PCNA的外表面具有多个功能表面，负责与多种因子相互作用。此外，两个侧面在功能上似乎是可区分的，这可能决定了PCNA沿DNA追踪的方向。
