DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI.
DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI.
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DOI：
10.1093/nar/gkp790
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发表时间：
2009-11
影响因子：
14.9
通讯作者：
Szczelkun MD
中科院分区：
文献类型：
--
作者：
Smith RM;Diffin FM;Savery NJ;Josephsen J;Szczelkun MD
LlaGI是天然存在于乳酸乳球菌质粒pEW104上的单多肽限制性修饰酶。cremoris W10。生物信息学分析表明，该酶含有mrr核酸内切酶、超家族2 DNA解旋酶和γ-家族腺嘌呤甲基转移酶的结构域。从重组克隆中表达和纯化了LlaGI，并对其性质进行了表征。鉴定出非对称识别序列5′-CTnGAyG-3′（其中n为A、G、C或T， y为C或T）。识别位点的甲基化仅发生在一条链上（5 ‘ -CrTCnAG-3 ’的非退化dA残基在N6位置被甲基化）。只有当两个头对头定向的、未甲基化的位点存在时，才会观察到在远处随机位点的双链DNA断裂；尾对尾或头对尾重复中的单个位点或成对只支持DNA切割活性。dsDNA核酸酶活性取决于ATP或dATP的存在。我们的结果与部分位点甲基化所必需的定向远程通信机制一致。在随附的手稿[Smith et al.(2009)]中，单多肽限制性修饰酶LlaGI是一个独立的分子马达，可易位DNA环]，我们证明了这种通信是通过一维DNA环易位进行的。基于这一数据以及所附的第三篇论文[Smith et al. (2009) An mr -family nuclease motif in the single polypeptide限制性修饰酶LlaGI]，我们提出LlaGI是限制性修饰酶的一个新子类的原型，命名为Type I SP （single polypeptide）。
LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a γ-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5′-CTnGAyG-3′ (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5′-CrTCnAG-3′ being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide).
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影响因子：
11.4
作者：
Firman, K;Szczelkun, MD
通讯作者：
Szczelkun, MD
影响因子：
64.8
作者：
MEISEL, A;BICKLE, TA;SCHROEDER, C
通讯作者：
SCHROEDER, C
影响因子：
14.9
作者：
Morgan, Richard D.;Bhatia, Tanya K.;Lovasco, Lindsay;Davis, Theodore B.
通讯作者：
Davis, Theodore B.
影响因子：
14.9
作者：
Roberts RJ;Vincze T;Posfai J;Macelis D
通讯作者：
Macelis D
影响因子：
11.4
作者：
MEISEL, A;MACKELDANZ, P;SCHROEDER, C
通讯作者：
SCHROEDER, C