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家族 1 casposase 介导的 Candidatus Nitrosopumilus koreensis AR1 序列特异性整合。

Sequence specific integration by the family 1 casposase from Candidatus Nitrosopumilus koreensis AR1.

机构信息

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing210009, China.

Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing210009, China.

出版信息

Nucleic Acids Res. 2021 Sep 27;49(17):9938-9952. doi: 10.1093/nar/gkab725.

DOI:10.1093/nar/gkab725
PMID:34428286
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8464041/
Abstract

Casposase, a homolog of Cas1 integrase, is encoded by a superfamily of mobile genetic elements known as casposons. While family 2 casposase has been well documented in both function and structure, little is known about the other three casposase families. Here, we studied the family 1 casposase lacking the helix-turn-helix (HTH) domain from Candidatus Nitrosopumilus koreensis AR1 (Ca. N. koreensis). The determinants for integration by Ca. N. koreensis casposase were extensively investigated, and it was found that a 13-bp target site duplication (TSD) sequence, a minimal 3-bp leader and three different nucleotides of the TSD sequences are indispensable for target specific integration. Significantly, the casposase can site-specifically integrate a broad range of terminal inverted repeat (TIR)-derived oligonucleotides ranging from 7-nt to ∼4000-bp, and various oligonucleotides lacking the 5'-TTCTA-3' motif at the 3' end of TIR sequence can be integrated efficiently. Furthermore, similar to some Cas1 homologs, the casposase utilizes a 5'-ATAA-3' motif in the TSD as a molecular ruler to dictate nucleophilic attack at 9-bp downstream of the end of the ruler during the spacer-side integration. By characterizing the family 1 Ca. N. koreensis casposase, we have extended our understanding on mechanistic similarities and evolutionary connections between casposons and the adaptation elements of CRISPR-Cas immunity.

摘要

Casposase 是一种与 Cas1 整合酶同源的蛋白，由一类被称为 casposons 的移动遗传元件所编码。虽然家族 2 的 casposase 在功能和结构方面已经得到了很好的研究，但对于其他三种 casposase 家族知之甚少。在这里，我们研究了来自 Candidatus Nitrosopumilus koreensis AR1（Ca. N. koreensis）的缺少螺旋-转角-螺旋（HTH）结构域的家族 1 casposase。我们广泛研究了 Ca. N. koreensis casposase 整合的决定因素，发现 13 个碱基对的靶位点重复（TSD）序列、最小的 3 个碱基对的前导序列和 TSD 序列的三个不同核苷酸对于靶特异性整合是必不可少的。重要的是，casposase 可以特异性地整合广泛的末端反向重复（TIR）衍生的寡核苷酸，范围从 7-nt 到约 4000-bp，并且各种缺乏 TIR 序列 3'端 5'-TTCTA-3' 基序的寡核苷酸也可以有效地被整合。此外，与一些 Cas1 同源物类似，casposase 在 TSD 中利用 5'-ATAA-3' 基序作为分子标尺，在间隔区侧整合过程中，在标尺末端下游 9 个碱基处决定亲核攻击。通过对家族 1 Ca. N. koreensis casposase 的特征分析，我们扩展了对 casposons 和 CRISPR-Cas 免疫的适应元件之间的机制相似性和进化联系的理解。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/a2f9ebc84d6b/gkab725fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/b14567571ce4/gkab725fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/4e7365e0fbed/gkab725fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/8ef4e450e3e4/gkab725fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/ee8472d8e00a/gkab725fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/f12d50ca738f/gkab725fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/1112b19f80e2/gkab725fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/a2f9ebc84d6b/gkab725fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/b14567571ce4/gkab725fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/4e7365e0fbed/gkab725fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/8ef4e450e3e4/gkab725fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/ee8472d8e00a/gkab725fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/f12d50ca738f/gkab725fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/1112b19f80e2/gkab725fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/336e/8464041/a2f9ebc84d6b/gkab725fig7.jpg

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CRISPR type II-A subgroups exhibit phylogenetically distinct mechanisms for prespacer insertion.CRISPR 类型 II-A 亚群表现出进化上不同的前间隔序列插入机制。
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