Roman Yaritzabel, Oshige Masahiko, Lee Young-Ju, Goodwin Kristie, Georgiadis Millie M, Hromas Robert A, Lee Suk-Hee
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Biochemistry. 2007 Oct 9;46(40):11369-76. doi: 10.1021/bi7005477. Epub 2007 Sep 18.
Metnase (SETMAR) is a SET and transposase fusion protein that promotes in vivo end joining activity and mediates genomic integration of foreign DNA. Recent studies showed that Metnase retained most of the transposase activities, including 5'-terminal inverted repeat (TIR)-specific binding and assembly of a paired end complex, and cleavage of the 5'-end of the TIR element. Here we show that R432 within the helix-turn-helix motif is critical for sequence-specific recognition, as the R432A mutation abolishes its TIR-specific DNA binding activity. Metnase possesses a unique DNA nicking and/or endonuclease activity that mediates cleavage of duplex DNA in the absence of the TIR sequence. While the HTH motif is essential for the Metnase-TIR interaction, it is not required for its DNA cleavage activity. The DDE-like motif is crucial for its DNA cleavage action as a point mutation at this motif (D483A) abolished its DNA cleavage activity. Together, our results suggest that Metnase's DNA cleavage activity, unlike those of other eukaryotic transposases, is not coupled to its sequence-specific DNA binding.
金属酶（SETMAR）是一种SET与转座酶融合蛋白，可促进体内末端连接活性并介导外源DNA的基因组整合。最近的研究表明，金属酶保留了大部分转座酶活性，包括5'-末端反向重复序列（TIR）特异性结合及双末端复合物的组装，以及TIR元件5'-末端的切割。在此我们表明，螺旋-转角-螺旋基序内的R432对于序列特异性识别至关重要，因为R432A突变消除了其TIR特异性DNA结合活性。金属酶具有独特的DNA切口和/或内切核酸酶活性，可在不存在TIR序列的情况下介导双链DNA的切割。虽然HTH基序对于金属酶与TIR的相互作用至关重要，但其DNA切割活性并不需要该基序。类DDE基序对于其DNA切割作用至关重要，因为该基序处的点突变（D483A）消除了其DNA切割活性。总之，我们的结果表明，与其他真核转座酶不同，金属酶的DNA切割活性与其序列特异性DNA结合不相关联。
