Mack A M, Crawford N M
Section of Cell and Developmental Biology, Division of Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0116, USA.
Plant Cell. 2001 Oct;13(10):2319-31. doi: 10.1105/tpc.010149.
The in vitro DNA binding activity of the Arabidopsis Tag1 transposase (TAG1) was characterized to determine the mechanism of DNA recognition. In addition to terminal inverted repeats, the Tag1 element contains four different subterminal repeats that flank a transcribed region encoding a 729-amino acid protein. A single site-specific DNA binding domain is located near the N terminus of TAG1, between residues 21 and 133. This domain binds specifically to the AAACCC and TGACCC subterminal repeats, found near the 5' and 3' ends of the element, respectively. The ACCC sequence within these repeats is critical for recognition because mutations at positions 3, 5, and 6 abolished binding, yet the first two bases also are important because substitutions at these positions decreased binding by up to 90%. Weak interaction also occurs with the terminal inverted repeats, but no binding was observed to the other two 3' subterminal repeat regions. Sequence analysis of the TAG1 DNA binding domain revealed a C(2)HC zinc finger motif. Tests for metal dependence showed that DNA binding activity was inhibited by divalent metal chelators and greatly enhanced by zinc. Furthermore, mutation of each cysteine residue predicted to be a metal ligand in the C(2)HC motif abolished DNA binding. Together, these data show that the DNA binding domain of TAG1 specifically binds to distinct subterminal repeats and contains a zinc finger.
对拟南芥Tag1转座酶（TAG1）的体外DNA结合活性进行了表征，以确定DNA识别机制。除了末端反向重复序列外，Tag1元件还包含四个不同的亚末端重复序列，它们位于一个编码729个氨基酸蛋白质的转录区域两侧。一个单一位点特异性DNA结合结构域位于TAG1的N末端附近，在第21和133位氨基酸残基之间。该结构域分别特异性结合元件5'和3'末端附近发现的AAACCC和TGACCC亚末端重复序列。这些重复序列中的ACCC序列对于识别至关重要，因为第3、5和6位的突变消除了结合，但前两个碱基也很重要，因为这些位置的取代使结合减少了高达90%。与末端反向重复序列也存在弱相互作用，但未观察到与其他两个3'亚末端重复区域的结合。对TAG1 DNA结合结构域的序列分析揭示了一个C(2)HC锌指基序。金属依赖性测试表明，DNA结合活性受到二价金属螯合剂的抑制，并被锌大大增强。此外，预测在C(2)HC基序中作为金属配体的每个半胱氨酸残基的突变都消除了DNA结合。总之，这些数据表明TAG1的DNA结合结构域特异性结合不同的亚末端重复序列，并包含一个锌指。
