The Type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites.
The Type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites.
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DOI：
10.1093/nar/gks1209
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发表时间：
2013-01
影响因子：
14.9
通讯作者：
Szczelkun MD
中科院分区：
文献类型：
--
作者：
Šišáková E;van Aelst K;Diffin FM;Szczelkun MD
ISP 型限制性修饰 (RM) 酶 LlaBIII 在质粒 pJW566 上编码，可以保护乳酸乳球菌菌株免受牛奶发酵中的噬菌体感染。它是一种单一多肽 RM 酶，包含 Mrr 核酸内切酶、DNA 解旋酶、腺嘌呤甲基转移酶和靶标识别结构域。 LlaBIII 在其前三个蛋白质结构域中与 ISP 型酶 LlaGI 具有 >95% 的氨基酸序列同源性。在这里，我们确定了 LlaBIII 的识别序列（5'-TnAGCC-3'，其中与下划线碱基互补的腺嘌呤被甲基化），并表征了其酶活性。 LlaBIII 与 LlaGI 具有相同的关键酶特征；即，三磷酸腺苷依赖性 DNA 易位（25°C 时约 309 bp/s）以及反向头对头重复中两个识别位点 DNA 切割的要求。然而，LlaBIII 需要 K+ 离子来防止非特异性 DNA 裂解，这些条件会影响 LlaGI 的易位和裂解特性。通过识别 LlaGI 或 LlaBIII 在不同缓冲条件下引入的非特异性 dsDNA 断裂的位置，我们验证了 ISP RM 型酶使用常见的易位碰撞机制来触发核酸内切酶活性。在他们喜欢的体外缓冲液中，LlaGI 和 LlaBIII 均产生以位点中间为中心的正态分布的随机切割位点。相比之下，K+ 离子中的 LlaGI 产生分布更均匀的裂解曲线。
The Type ISP Restriction–Modification (RM) enzyme LlaBIII is encoded on plasmid pJW566 and can protect Lactococcus lactis strains against bacteriophage infections in milk fermentations. It is a single polypeptide RM enzyme comprising Mrr endonuclease, DNA helicase, adenine methyltransferase and target-recognition domains. LlaBIII shares >95% amino acid sequence homology across its first three protein domains with the Type ISP enzyme LlaGI. Here, we determine the recognition sequence of LlaBIII (5′-TnAGCC-3′, where the adenine complementary to the underlined base is methylated), and characterize its enzyme activities. LlaBIII shares key enzymatic features with LlaGI; namely, adenosine triphosphate-dependent DNA translocation (∼309 bp/s at 25°C) and a requirement for DNA cleavage of two recognition sites in an inverted head-to-head repeat. However, LlaBIII requires K+ ions to prevent non-specific DNA cleavage, conditions which affect the translocation and cleavage properties of LlaGI. By identifying the locations of the non-specific dsDNA breaks introduced by LlaGI or LlaBIII under different buffer conditions, we validate that the Type ISP RM enzymes use a common translocation–collision mechanism to trigger endonuclease activity. In their favoured in vitro buffer, both LlaGI and LlaBIII produce a normal distribution of random cleavage loci centred midway between the sites. In contrast, LlaGI in K+ ions produces a far more distributive cleavage profile.
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影响因子：
14.9
作者：
Smith RM;Josephsen J;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.
DOI：
10.1107/s1744309111028041
发表时间：
2011-10-01
影响因子：
0.9
作者：
Callahan, Scott J.;Morgan, Richard D.;Aggarwal, Aneel K.
通讯作者：
Aggarwal, Aneel K.
影响因子：
5.6
作者：
McClelland, SE;Dryden, DTF;Szczelkun, MD
通讯作者：
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