Iida Tetsuo, Suetake Isao, Tajima Shoji, Morioka Hiroshi, Ohta Satoshi, Obuse Chikashi, Tsurimoto Toshiki
Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101, Japan.
Genes Cells. 2002 Oct;7(10):997-1007. doi: 10.1046/j.1365-2443.2002.00584.x.
Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase delta. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied.
Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA.
These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp.
增殖细胞核抗原（PCNA）是一种环状蛋白，作为DNA聚合酶δ的持续合成因子为人所知。除了这一作用外，PCNA还与许多其他蛋白相互作用，以增加它们在复制DNA位点的局部浓度。DNA胞嘧啶甲基转移酶1（Dnmt1）通过在DNA复制后完成半甲基化DNA的甲基化来保留表观遗传信号，先前的一项研究已表明它是这些PCNA结合蛋白之一。然而，它们相互作用的分子机制和功能意义尚未得到研究。
通过PCNA亲和层析可轻易从核提取物中分离出Dnmt1。对这两种蛋白之间相互作用的研究表明，Dnmt1的N端区域含有典型的PCNA结合基序，具有核心PCNA结合活性，而该蛋白的其余部分对Dnmt1与PCNA的相互作用产生负面影响。Dnmt1对与PCNA结合的DNA的亲和力比对游离DNA的亲和力高得多。此外，以半甲基化DNA为底物的DNA甲基化分析表明，与游离DNA相比，Dnmt1对PCNA钳夹结合的DNA甲基化效率更高。
这些结果提供了首个生化证据，即PCNA与Dnmt1之间的物理相互作用促进了新复制DNA的甲基化，PCNA作为一种功能性钳夹仍与新复制DNA结合。
