Chen Qiujia, Georgiadis Millie
Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.
Acta Crystallogr F Struct Biol Commun. 2016 Sep;72(Pt 9):713-9. doi: 10.1107/S2053230X16012723. Epub 2016 Aug 26.
Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of the Hsmar1 transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestral Hsmar1 terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD-DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.
转座元件在包括人类在内的所有高等真核生物新基因的产生过程中发挥了关键作用。尽管嵌合融合蛋白SETMAR不再作为转座酶发挥作用，但它同时包含Hsmar1转座酶的DNA结合结构域（DBD）和催化结构域。DBD的氨基酸序列在五千万年里几乎没有变化，因此，SETMAR保留了其与祖先Hsmar1末端反向重复序列（TIR）的序列特异性结合。因此，SETMAR的DNA结合活性可能具有重要的生物学功能。为了确定SETMAR识别TIR DNA的结构基础，本文报道了含TIR寡核苷酸和SETMAR DBD变体的设计、DBD-DNA复合物的结晶、相位测定策略和初步相位测定实验。一个意外的发现是，含有两个溴脱氧尿苷替代胸腺嘧啶的寡核苷酸与SETMAR形成的复合物晶体质量比天然对应物更好。
