The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity.
The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity.
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来自吡啶家的CRISPR相关的CAS4蛋白PCAL_0546包含[2FE-2S]簇：晶体结构和核酸酶活性。
DOI：
10.1093/nar/gku797
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发表时间：
2014
影响因子：
14.9
通讯作者：
Yakunin AF
中科院分区：
文献类型：
--
作者：
Lemak S;Nocek B;Beloglazova N;Skarina T;Flick R;Brown G;Joachimiak A;Savchenko A;Yakunin AF
Cas4核酸酶构成了CRISPR （Clustered Regularly Interspaced Short Palindromic Repeats）相关蛋白的核心家族，但对其结构和活性知之甚少。本文报道了caldifontis焦杆菌Cas4蛋白Pcal_0546的晶体结构，该蛋白具有recb样折叠和一个由四个保守的Cys残基协调的[2Fe-2S]簇。Pcal_0546对ssDNA底物表现出金属依赖的5 ‘至3 ’外切酶活性，而来自Sulfolobus solfataricus的Cas4蛋白SSO1391可以在5 ‘至3 ’和3 ‘至5 ’方向上切割ssDNA。Pcal_0546的活性位点含有一个由Asp123、Glu136、His146侧链和Ile137主链羰基配位的结合金属离子。Pcal_0546和SSO1391的定点突变表明，RecB基序II、III和QhXXY的残基对核酸酶活性至关重要，而保守的Cys残基的突变导致铁硫簇的丢失，但对DNA切割没有影响。我们的研究结果揭示了Cas4核酸酶的生化多样性，它们可以具有不同的寡聚状态，含有[4Fe-4S]或[2Fe-2S]簇，并在不同方向上切割单链DNA，产生单链DNA悬垂，这是合成新的CRISPR间隔物的潜在中间体。
Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5′ to 3′ exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5′ to 3′ and 3′ to 5′ directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.
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DOI：
10.1126/science.1165771
发表时间：
2008-12-19
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Marraffini LA;Sontheimer EJ
通讯作者：
Sontheimer EJ
影响因子：
5.2
作者：
Bernick DL;Cox CL;Dennis PP;Lowe TM
通讯作者：
Lowe TM
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.9
作者：
Mitou, G;Higgins, C;Meyer, J
通讯作者：
Meyer, J
影响因子：
11.4
作者：
Beloglazova, Natalia;Petit, Pierre;Yakunin, Alexander F.
通讯作者：
Yakunin, Alexander F.