Chen L F, Ito K, Murakami Y, Ito Y
Department of Viral Oncology, Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606, Japan.
Mol Cell Biol. 1998 Jul;18(7):4165-76. doi: 10.1128/MCB.18.7.4165.
The nuclear matrix is thought to play an important role in the DNA replication of eukaryotic cells, although direct evidence for such a role is still lacking. A nuclear matrix-associated transcription factor, polyomavirus (Py) enhancer binding protein 2alphaB1 (PEBP2alphaB1) (AML1/Cbfa2), was found to stimulate Py replication through its cognate binding site. The minimal replication activation domain (RAD) was identified between amino acid (aa) 302 and aa 371 by using a fusion protein containing the GAL4 DNA binding domain (GAL4-RAD). In addition, the region showed affinity for the nuclear matrix and, on the basis of competition studies, binding activity for one or more proteins involved in the initiation of Py DNA replication. A leukemogenic chimeric protein, AML1/ETO(MTG8), which does not contain this region of PEBP2alphaB1/AML1, was also localized in the nuclear matrix fraction and competed for nuclear matrix association with PEBP2alphaB1 and GAL4-RAD. Moreover, AML1/ETO inhibited Py DNA replication stimulated by PEBP2alphaB1 and GAL4-RAD. The inhibition was specific for replication mediated by PEBP2alphaB1 and GAL4-RAD, and proportional to the degree of loss of these activators from the nuclear matrix, suggesting a requirement for nuclear matrix targeting in the stimulation of Py DNA replication by RAD. These results are the first to suggest a molecular link between the initiation of DNA replication and the nuclear matrix compartment.
尽管仍缺乏真核细胞DNA复制中核基质发挥这一作用的直接证据，但人们认为核基质在真核细胞的DNA复制中起重要作用。一种与核基质相关的转录因子，多瘤病毒（Py）增强子结合蛋白2αB1（PEBP2αB1）（AML1/Cbfa2），被发现可通过其同源结合位点刺激Py复制。通过使用含有GAL4 DNA结合结构域的融合蛋白（GAL4-RAD），在氨基酸（aa）302和aa 371之间鉴定出最小复制激活结构域（RAD）。此外，该区域对核基质具有亲和力，并且基于竞争研究，对参与Py DNA复制起始的一种或多种蛋白质具有结合活性。一种致白血病嵌合蛋白AML1/ETO（MTG8），它不包含PEBP2αB1/AML1的这一区域，也定位于核基质组分中，并与PEBP2αB1和GAL4-RAD竞争核基质结合。此外，AML1/ETO抑制由PEBP2αB1和GAL4-RAD刺激的Py DNA复制。这种抑制对由PEBP2αB1和GAL4-RAD介导的复制具有特异性，并且与这些激活剂从核基质中丧失的程度成比例，这表明RAD刺激Py DNA复制需要靶向核基质。这些结果首次表明DNA复制起始与核基质区室之间存在分子联系。
