Kuduvalli Prasad N, Mitra Rupak, Craig Nancy L
The Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine Baltimore, MD 21205, USA.
Nucleic Acids Res. 2005 Feb 8;33(3):857-63. doi: 10.1093/nar/gki227. Print 2005.
The bacterial transposon, Tn7, inserts into a single site in the Escherichia coli chromosome termed attTn7 via the sequence-specific DNA binding of the target selector protein, TnsD. The target DNA sequence required for Tn7 transposition is located within the C-terminus of the glucosamine synthetase (glmS) gene, which is an essential, highly conserved gene found ubiquitously from bacteria to humans. Here, we show that Tn7 can transpose in vitro adjacent to two potential targets in the human genome: the gfpt-1 and gfpt-2 sequences, the human analogs of glmS. The frequency of transposition adjacent to the human gfpt-1 target is comparable with the E.coli glmS target; the human gfpt-2 target shows reduced transposition. The binding of TnsD to these sequences mirrors the transposition activity. In contrast to the human gfpt sequences, Tn7 does not transpose adjacent to the gfa-1 sequence, the glmS analog in Saccharomyces cerevisiae. We also report that a nucleosome core particle assembled on the human gfpt-1 sequence reduces Tn7 transposition by likely impairing the accessibility of target DNA to the Tns proteins. We discuss the implications of these findings for the potential use of Tn7 as a site-specific DNA delivery agent for gene therapy.
细菌转座子Tn7通过靶标选择蛋白TnsD的序列特异性DNA结合，插入大肠杆菌染色体中一个称为attTn7的单一位点。Tn7转座所需的靶DNA序列位于氨基葡萄糖合成酶（glmS）基因的C末端，该基因是一个从细菌到人类普遍存在的必需且高度保守的基因。在此，我们表明Tn7可在体外转座至人类基因组中的两个潜在靶标附近：gfpt-1和gfpt-2序列，即人类的glmS类似物。与人类gfpt-1靶标相邻的转座频率与大肠杆菌glmS靶标相当；人类gfpt-2靶标显示转座减少。TnsD与这些序列的结合反映了转座活性。与人类gfpt序列不同，Tn7不会在酿酒酵母中glmS的类似物gfa-1序列附近转座。我们还报告称，组装在人类gfpt-1序列上的核小体核心颗粒可能通过损害靶DNA对Tns蛋白的可及性来降低Tn7转座。我们讨论了这些发现对于将Tn7用作基因治疗的位点特异性DNA递送剂的潜在用途的意义。
