Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition.
Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition.
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DOI：
10.7554/elife.08716
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发表时间：
2015-08-18
期刊：
影响因子：
7.7
通讯作者：
White MF
中科院分区：
文献类型：
--
作者：
Rollie C;Schneider S;Brinkmann AS;Bolt EL;White MF
适应性原核免疫系统CRISPR-Cas提供了RNA介导的保护免受入侵的遗传元件。该系统的基本基础是捕获小片段外源DNA以在CRISPR基因座处掺入基因组的能力，这一过程称为适应，这取决于Cas 1和Cas 2蛋白。我们证明，Cas 1催化一个有效的酯交换反应的分支的DNA基板，这代表了逆或解体反应。来自大肠杆菌和硫磺硫化叶菌的Cas 1都显示出序列特异性活性，明显偏好CRISPR基因座的前导重复序列1边界处整合位点侧翼的核苷酸。Cas 2不是该活性所必需的，并且不影响特异性。这表明Cas 1的固有序列特异性是适应过程的主要决定因素。http://dx.doi.org/10.7554/eLife.08716.001在大多数动物中，适应性免疫系统产生专门的细胞，这些细胞能够有效地抵抗任何入侵的病毒或其他病原体。细菌（以及另一组称为古细菌的单细胞生物）也有一种适应性免疫系统，称为CRISPR-Cas，可以对抗病毒入侵者。该系统基于微生物DNA的CRISPR部分，其中包含由“间隔区”DNA短片段分隔的重复DNA序列。当病毒侵入细胞时，一些病毒DNA作为间隔区被整合到CRISPR中。这个过程被称为适应。然后，CRISPR相关蛋白（或“Cas”蛋白）使用该间隔区识别并攻击随后遇到的任何匹配的入侵者DNA。在适应过程中，间隔区如何插入CRISPR阵列的正确位置仍然知之甚少。然而，已知两种名为Cas 1和Cas 2的CRISPR蛋白在此过程中发挥重要作用。Rollie等人从细菌细胞（大肠杆菌）和古细菌物种（Sulfolobus solfataricus）中提取了Cas 1蛋白，并在实验室中将其添加到分支DNA结构中。这些实验表明，来自这两种生物的Cas 1可以将DNA分解成更小的片段。另一方面，Cas 2对于该过程不是必需的。这种“分解”反应是适应的“整合”步骤的逆过程，其中CRISPR蛋白将入侵者DNA插入CRISPR阵列中。Rollie等人还发现，Cas 1进行的解体反应发生在特定的DNA序列上，这些序列也是Cas 1在适应过程中插入间隔区DNA的位点。因此，通过检查衰变反应，可以推导出积分步骤的许多细节。总体而言，Rollie等人表明，Cas 1的选择在将适应过程限制在特定DNA位点方面起着重要作用。下一步将是使用解体反应来检查Cas 1执行的DNA结合和操作步骤，作为其在CRISPR系统适应中的作用的一部分。DOI：http://dx.doi.org/10.7554/eLife.08716.002网站
The adaptive prokaryotic immune system CRISPR-Cas provides RNA-mediated protection from invading genetic elements. The fundamental basis of the system is the ability to capture small pieces of foreign DNA for incorporation into the genome at the CRISPR locus, a process known as Adaptation, which is dependent on the Cas1 and Cas2 proteins. We demonstrate that Cas1 catalyses an efficient trans-esterification reaction on branched DNA substrates, which represents the reverse- or disintegration reaction. Cas1 from both Escherichia coli and Sulfolobus solfataricus display sequence specific activity, with a clear preference for the nucleotides flanking the integration site at the leader-repeat 1 boundary of the CRISPR locus. Cas2 is not required for this activity and does not influence the specificity. This suggests that the inherent sequence specificity of Cas1 is a major determinant of the adaptation process. DOI: http://dx.doi.org/10.7554/eLife.08716.001 In most animals, the adaptive immune system creates specialized cells that adapt to efficiently fight off any viruses or other pathogens that have invaded. Bacteria (and another group of single-celled organisms called archaea) also have an adaptive immune system, known as CRISPR-Cas, that combats viral invaders. This system is based on sections of the microbes' DNA called CRISPRs, which contain repetitive DNA sequences that are separated by short segments of ‘spacer’ DNA. When a virus invades the cell, some viral DNA is incorporated into the CRISPR as a spacer. This process is known as adaptation. CRISPR-associated proteins (or ‘Cas’ proteins) then use this spacer to recognize and mount an attack on any matching invader DNA that is later encountered. Exactly how a spacer is inserted into the correct position in the CRISPR array during adaptation remains poorly understood. However, it is known that two CRISPR proteins called Cas1 and Cas2 play essential roles in this process. Rollie et al. took Cas1 proteins from a bacterial cell (Escherichia coli) and an archaeal species (Sulfolobus solfataricus) and added them to branched DNA structures in the laboratory. These experiments revealed that Cas1 from both organisms can break the DNA down into smaller pieces. Cas2, on the other hand, is not required for this process. This ‘disintegration’ reaction is the reverse process of the ‘integration’ step of adaptation where the CRISPR proteins insert the invader DNA into the CRISPR array. Rollie et al. also found that the disintegration reaction performed by Cas1 takes place on specific DNA sequences, which are also the sites where Cas1 inserts the spacer DNA during adaptation. Therefore, by examining the disintegration reaction, many of the details of the integration step can be deduced. Overall, Rollie et al. show that selection by Cas1 plays an important role in restricting the adaptation process to particular DNA sites. The next step will be to use the disintegration reaction to examine the DNA binding and manipulation steps performed by Cas1 as part of its role in the adaptation of the CRISPR system. DOI: http://dx.doi.org/10.7554/eLife.08716.002