I-TEVI, THE ENDONUCLEASE ENCODED BY THE MOBILE TD INTRON, RECOGNIZES BINDING AND CLEAVAGE DOMAINS ON ITS DNA TARGET
I-TEVI, THE ENDONUCLEASE ENCODED BY THE MOBILE TD INTRON, RECOGNIZES BINDING AND CLEAVAGE DOMAINS ON ITS DNA TARGET
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DOI：
10.1073/pnas.88.17.7719
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发表时间：
1991-09-01
影响因子：
11.1
通讯作者：
BELFORT, M
中科院分区：
文献类型：
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作者：
BELLPEDERSEN, D;QUIRK, SM;BELFORT, M
噬菌体T4 td内含子的迁移率取决于内含子编码的内切核酸酶（I-TevI）的活性，该内切核酸酶切割同源无内含子（DELTA-In）靶基因。双链断裂引发重组事件，导致内含子转移。我们先前舍入了I-TevI切割内含子插入位点上游23-26个核苷酸的td-DELTA-In靶DNA。DNA酶I足迹实验和凝胶迁移分析表明，I-TevI使周围的内含子插入位点的主要接触。跨越插入位点但缺乏切割位点的合成DNA双链体显示特异性结合I-TevI，并且当克隆时，直接切割成载体序列。克隆的双链体的行为和缺失和插入突变体的行为支持的插入位点周围的序列在指导I-TevI结合，赋予切割能力，并确定切割极性的主要作用。另一方面，切割位点周围的序列显示影响切割效率和切割位点选择。裂解位点序列在确定裂解距离中的作用反对I-TevI裂解的严格“标尺”机制。这种不寻常的类型的核酸内切酶识别的归巢位点的复杂性被认为是在内含子传播的背景下。
Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (DELTA-In) target gene. The double-strand break initiates a recombination event that leads to intron transfer. We round previously that I-TevI cleaves td-DELTA-In target DNA 23-26 nucleotides upstream of the intron insertion site. DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion site. A synthetic DNA duplex spanning the insertion site but lacking the cleavage site was shown to bind I-TevI specifically, and when cloned, to direct cleavage into vector sequences. The behavior of the cloned duplex and that of deletion and insertion mutants support a primary role for sequences surrounding the insertion site in directing I-TevI binding, conferring cleavage ability, and determining cleavage polarity. On the other hand, sequences around the cleavage site were shown to influence cleavage efficiency and cut-site selection. The role of cleavage-site sequences in determining cleavage distance argues against a strict "ruler" mechanism for cleavage by I-TevI. The complex nature of the homing site recognized by this unusual type of endonuclease is considered in the context of intron spread.