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首页> 外文期刊>Nucleic Acids Research >Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis
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Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

机译：生化分析来自噬菌体T4的内切核酸酶IV的底物特异性和序列偏好性，噬菌体T4是dC取代的T4 DNA合成受限的dC特异性内切核酸酶

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摘要

Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5' phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5'-dTdCdA-3' but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5'-dTdCdA-3', which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA.
机译：由噬菌体T4的denB编码的核酸内切酶IV与宿主大肠杆菌中含脱氧胞苷（dC）的DNA的限制有关。该酶是利用无小麦生殖细胞的蛋白质合成系统合成的，具有在大肠杆菌细胞中表达的致命作用，并纯化至均一。纯化的酶对单链（ss）DNA和变性的dC取代的T4基因组双链（ds）DNA表现出高活性，但对含有糖基化的脱氧羟甲基甲基胞苷的dsDNA，ssRNA或变性的T4基因组dsDNA没有活性。 Endo IV活性的表征表明，该酶催化ssDNA中dC的5'磷酸二酯键的特异性内切核酸酶裂解，其效率明显取决于周围的核苷酸序列。该酶优先靶向5'-dTdCdA-3'，但可耐受该三核苷酸序列侧翼的各个核苷酸的各种组合。这些结果表明，Endo IV优先识别含有5'-dTdCdA-3'的短核苷酸序列，这可能是由于该酶对ssDNA的消化作用有限，并且可能部分归因于denB缺乏以稳定合成SnDNA的原因。 dC取代的T4基因组DNA。

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