Gong Z, Hew C L
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Biochemistry. 1994 Dec 20;33(50):15149-58. doi: 10.1021/bi00254a026.
LIM homeodomain proteins are a family of recently characterized proteins which contain, in addition to a homeodomain, two tandem repeats of conserved Cys-His motifs termed as LIM domains. We have recently isolated several clones from a chinook salmon pituitary cDNA library that encode two novel LIM homeodomain proteins, Isl-2 and Isl-3, which are structurally related to rat Isl-1. In the present study, we used the salmon Isl-2 to determine the role of LIM domains in DNA binding. Several glutathione S-transferase (GST) fusion proteins containing either full length Isl-2 or various portions of this protein were expressed in bacteria. Zinc blot analysis reveals that the LIM domains produced in bacteria are capable of binding zinc. Gel shift analysis indicates that all homeodomain-containing fusion proteins are able to bind to a TAAT target sequence while the fusion proteins containing only the LIM domain are not. In contrast to a previous observation that the LIM domains of rat Isl-1 have an inhibitory role in DNA binding, full length salmon Isl-2 containing both the LIM domains and a homeodomain can bind to a TAAT target sequence. To further examine the role of LIM domains in DNA binding, several GST fusion proteins were used to select specific target DNA sequences from a pool of randomly incorporated oligonucleotides. Specific target DNAs were selected by fusion proteins containing the homeodomain or the full length Isl-2, but not by LIM domain only fusion proteins, indicating that the LIM domain alone is not involved in DNA binding. The selected target DNAs were cloned and sequenced. They revealed two classes of consensus, C/TTAATG/TG/A and C/TTAAGTG, for both the homeodomain and full length Isl-2. The two classes of consensus competed with each other for binding to the homeodomain. The equilibrium dissociation constants for DNA binding, estimated by Scatchard analysis, were similar for the homeodomain and full length Isl-2.(ABSTRACT TRUNCATED AT 400 WORDS)
LIM 同源结构域蛋白是一类最近才被鉴定的蛋白质家族，除了一个同源结构域外，还包含两个被称为 LIM 结构域的保守半胱氨酸 - 组氨酸基序的串联重复序列。我们最近从奇努克鲑鱼垂体 cDNA 文库中分离出了几个克隆，它们编码两种新的 LIM 同源结构域蛋白，即 Isl - 2 和 Isl - 3，它们在结构上与大鼠 Isl - 1 相关。在本研究中，我们使用鲑鱼 Isl - 2 来确定 LIM 结构域在 DNA 结合中的作用。几种含有全长 Isl - 2 或该蛋白不同部分的谷胱甘肽 S - 转移酶（GST）融合蛋白在细菌中表达。锌印迹分析表明，在细菌中产生的 LIM 结构域能够结合锌。凝胶迁移分析表明，所有含同源结构域的融合蛋白都能够结合到 TAAT 靶序列上，而仅含 LIM 结构域的融合蛋白则不能。与之前观察到的大鼠 Isl - 1 的 LIM 结构域在 DNA 结合中具有抑制作用相反，同时包含 LIM 结构域和同源结构域的全长鲑鱼 Isl - 2 能够结合到 TAAT 靶序列上。为了进一步研究 LIM 结构域在 DNA 结合中的作用，使用了几种 GST 融合蛋白从一组随机掺入的寡核苷酸中选择特定的靶 DNA 序列。特定的靶 DNA 是由含同源结构域或全长 Isl - 2 的融合蛋白选择的，而仅含 LIM 结构域的融合蛋白则不能，这表明单独的 LIM 结构域不参与 DNA 结合。所选的靶 DNA 被克隆并测序。它们揭示了同源结构域和全长 Isl - 2 的两类共有序列，即 C/TTAATG/TG/A 和 C/TTAAGTG。这两类共有序列相互竞争与同源结构域的结合。通过 Scatchard 分析估计的 DNA 结合平衡解离常数，同源结构域和全长 Isl - 2 相似。（摘要截短至 400 字）
