Wahls W P, Moore P D
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Somat Cell Mol Genet. 1998 Jan;24(1):41-51. doi: 10.1007/BF02677494.
Hypervariable minisatellite DNA repeats are found at tens of thousands of loci in the mammalian genome. These sequences stimulate homologous recombination in mammalian cells [Cell 60:95-103]. To test the hypothesis that protein-DNA interaction is required for hotspot function in vivo, we determined whether a second protein binding nearby could abolish hotspot activity. Intermolecular recombination between pairs of plasmid substrates was measured in the presence or absence of the cis-acting recombination hotspot and in the presence or absence of the second trans-acting DNA binding protein. Minisatellite DNA had hotspot activity in two cell lines, but lacked hotspot activity in two closely related cell lines expressing a site-specific helicase that bound to DNA adjacent to the hotspot. Suppression of hotspot function occurred for both replicating and non-replicating recombination substrates. These results indicate that hotspot activity in vivo requires site occupancy by minisatellite DNA binding proteins.
高变微卫星DNA重复序列存在于哺乳动物基因组的数万个位点上。这些序列可刺激哺乳动物细胞中的同源重组[《细胞》60:95 - 103]。为了验证体内热点功能需要蛋白质 - DNA相互作用这一假说，我们确定了附近的第二种蛋白质结合是否会消除热点活性。在存在或不存在顺式作用重组热点以及存在或不存在第二种反式作用DNA结合蛋白的情况下，测量了质粒底物对之间的分子间重组。微卫星DNA在两种细胞系中具有热点活性，但在两种表达与热点相邻DNA结合的位点特异性解旋酶的密切相关细胞系中缺乏热点活性。对于复制型和非复制型重组底物，热点功能均受到抑制。这些结果表明，体内热点活性需要微卫星DNA结合蛋白占据位点。
