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首页> 外文期刊>Journal of Molecular Biology >Characterization of the C-Terminal DNA-binding/DNA Endonuclease Region of a Group II Intron-encoded Protein.
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Characterization of the C-Terminal DNA-binding/DNA Endonuclease Region of a Group II Intron-encoded Protein.

机译：II组内含子编码蛋白的C端DNA结合/ DNA核酸内切酶区域的表征。

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摘要

Group II intron retrohoming occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase uses a C-terminal DNA endonuclease activity to cleave the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. Here, we characterized the C-terminal DNA-binding/DNA endonuclease region of the LtrA protein encoded by the Lactococcus lactis Ll.LtrB intron. This C-terminal region consists of an upstream segment that contributes to DNA binding, followed by a DNA endonuclease domain that contains conserved sequence motifs characteristic of H-N-H DNA endonucleases, interspersed with two pairs of conserved cysteine residues. Atomic emission spectroscopy of wild-type and mutant LtrA proteins showed that the DNA endonuclease domain contains a single tightly bound Mg(2+) ion at the H-N-H active site. Although the conserved cysteine residue pairs could potentially bind Zn(2+), the purified LtrA protein is active despite the presence of only sub-stoichiometric amounts of Zn(2+), and the addition of exogenous Zn(2+) inhibits the DNA endonuclease activity. Multiple sequence alignments identified features of the DNA-binding region and DNA endonuclease domain that are conserved in LtrA and related group II intron proteins, and their functional importance was demonstrated by unigenic evolution analysis and biochemical assays of mutant LtrA protein with alterations in key amino acid residues. Notably, deletion of the DNA endonuclease domain or mutations in its conserved sequence motifs strongly inhibit reverse transcriptase activity, as well as bottom-strand cleavage, while retaining other activities of the LtrA protein. A UV-cross-linking assay showed that these DNA endonuclease domain mutations do not block DNA primer binding and thus likely inhibit reverse transcriptase activity either by affecting the positioning of the primer or the conformation of the reverse transcriptase domain.
机译：第II组内含子逆向归巢是通过一种机制进行的，其中内含子RNA反向直接剪接成双链DNA靶位点的一条链，而内含子编码的逆转录酶利用C端DNA核酸内切酶活性来切割相反的链，然后使用裂解的3'端作为引物，用于插入的内含子RNA的逆转录。在这里，我们表征了由乳酸乳球菌Ll.LtrB内含子编码的LtrA蛋白的C端DNA结合/ DNA核酸内切酶区域。该C末端区域由有助于DNA结合的上游区段组成，随后是DNA内切核酸酶结构域，其包含H-N-H DNA内切核酸酶特征性的保守序列基序，并散布有两对保守的半胱氨酸残基。野生型和突变LtrA蛋白的原子发射光谱表明，DNA核酸内切酶结构域在H-N-H活性位点包含一个紧密结合的Mg（2+）离子。尽管保守的半胱氨酸残基对可能会结合Zn（2+），尽管仅存在亚化学计量的Zn（2+），但纯化的LtrA蛋白还是有活性的，外源Zn（2+）的加入会抑制DNA核酸内切酶活性。多个序列比对鉴定了LtrA和相关的II组内含子蛋白中保守的DNA结合区和DNA核酸内切酶结构域的特征，并且通过单基因进化分析和突变LtrA蛋白的生化分析证明了关键氨基酸的改变，证明了其功能重要性残留物。值得注意的是，DNA核酸内切酶结构域的缺失或其保守序列基序中的突变强烈抑制了逆转录酶活性以及底链裂解，同时保留了LtrA蛋白的其他活性。紫外交联测定表明，这些DNA核酸内切酶结构域突变不会阻断DNA引物结合，因此可能通过影响引物的位置或逆转录酶结构域的构象而抑制逆转录酶活性。

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