Background

Restriction endonucleases belong to prokaryotic restriction-modification systems, that protect host cells from invading DNA. Type II restriction endonucleases recognize short 4–8 bp sequences in the target DNA and cut both DNA strands producing double strand breaks. Type II restriction endonuclease Kpn2I cleaves 5′-T/CCGGA DNA sequence (“/” marks the cleavage position). Analysis of protein sequences suggested that Kpn2I belongs to the CCGG-family, which contains ten enzymes that recognize diverse nucleotides outside the conserved 5′-CCGG core and share similar motifs for the 5′-CCGG recognition and cleavage.

Methods

We solved a crystal structure of Kpn2I in a DNA-free form at 2.88 Å resolution. From the crystal structure we predicted active center and DNA recognition residues and tested them by mutational analysis. We estimated oligomeric state of Kpn2I by SEC-MALS and performed plasmid DNA cleavage assay to elucidate DNA cleavage mechanism.

Results

Structure comparison confirmed that Kpn2I shares a conserved active site and structural determinants for the 5′-CCGG tetranucleotide recognition with other restriction endonucleases of the CCGG-family. Guided by structural similarity between Kpn2I and the CCGG-family restriction endonucleases PfoI and AgeI, Kpn2I residues involved in the outer base pair recognition were proposed.

Conclusions

Kpn2I is an orthodox Type IIP restriction endonuclease, which acts as a dimer. Kpn2I shares structural similarity to the CCGG-family restriction endonucleases PfoI, AgeI and PspGI.

General significance

The Kpn2I structure concluded the studies of the CCGG-family, covering detailed structural and biochemical characterization of eleven restriction enzymes and their complexes with DNA.

中文翻译：

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CCGG家族限制性内切酶Kpn2I的晶体结构

背景

限制性核酸内切酶属于原核限制性修饰系统，可保护宿主细胞免于入侵 DNA。II 型限制性内切核酸酶识别目标 DNA 中的 4-8 bp 短序列，并切割两条 DNA 链，产生双链断裂。II 型限制性内切核酸酶 Kpn2I 切割 5'-T/CCGGA DNA 序列（“/”标记切割位置）。蛋白质序列分析表明，Kpn2I 属于 CCGG 家族，该家族包含 10 种酶，可识别保守的 5'-CCGG 核心之外的不同核苷酸，并共享相似的 5'-CCGG 识别和切割基序。

方法

我们以 2.88 Å 的分辨率解析了无 DNA 形式的 Kpn2I 晶体结构。根据晶体结构，我们预测了活性中心和 DNA 识别残基，并通过突变分析对其进行了测试。我们通过 SEC-MALS 估计了 Kpn2I 的寡聚状态，并进行了质粒 DNA 切割测定以阐明 DNA 切割机制。

结果

结构比较证实 Kpn2I 与 CCGG 家族的其他限制性内切核酸酶共享 5'-CCGG 四核苷酸识别的保守活性位点和结构决定簇。以 Kpn2I 和 CCGG 家族限制性内切核酸酶 PfoI 和 AgeI 之间的结构相似性为指导，提出了参与外部碱基对识别的 Kpn2I 残基。

结论

Kpn2I 是一种正统的 IIP 型限制性内切核酸酶，它充当二聚体。Kpn2I 与 CCGG 家族限制性内切核酸酶 PfoI、AgeI 和 PspGI 具有结构相似性。

一般意义

Kpn2I 结构结束了 CCGG 家族的研究，涵盖了 11 种限制酶及其与 DNA 复合物的详细结构和生化特征。