BACKGROUND Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC. RESULTS The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360), cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases. CONCLUSION We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.

中文翻译：

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BsaHI 限制修饰系统：保守基序的克隆、测序和分析。

背景技术限制酶和修饰酶通常识别长度为2至8个碱基的短DNA序列。了解这种识别的机制代表了我们开始为 BsaHI 限制修饰系统解决的重大挑战，该系统识别六碱基序列 GRCGYC。结果 BsaHI 甲基转移酶 bsaHIM 和限制性内切核酸酶 bsaHIR 的 DNA 序列已经确定（GenBank 登录号 #EU386360），并在大肠杆菌中克隆和表达。限制性核酸内切酶和甲基转移酶与一组其他 6 种酶（包括限制性修饰系统 HgiDI 和 HgiGI 以及推定的 HindVP、NlaCORFDP、NpuORFC228P 和 SplZORFNP 限制性修饰系统）具有显着的相似性。这些同源物的序列比对表明它们的氨基酸序列在很大程度上是保守的，并突出了几个感兴趣的基序。我们在 bsaHIR 基因的 C 末端靶向一种这样的保守基序，读取 SPERFD。这些氨基酸的突变分析表明该基序对酶活性至关重要。甲基转移酶基因的序列比对揭示了目标识别域内的一个短基序，该基序在识别相同序列的酶之间是保守的。因此，该基序可用作诊断工具来定义胞嘧啶 C5 甲基转移酶的识别序列。结论 我们已经克隆并测序了 BsaHI 限制性和修饰酶。我们已经确定了对其活性至关重要的 R. BsaHI 酶区域。