摘要

革兰氏阴性菌适应性的主要工具是称为整合子的功能性遗传平台。整合子在独特的重组过程中捕获和重排无启动子基因盒，涉及识别折叠的单链DNA发夹 - 所谓的attC位点 - 强烈偏爱attC底链。虽然已经确定了结构要素以促进这种偏好，但其机械作用仍然不完整。在这里，我们使用高分辨率单分子光学镊子（OT）来表征由attC底部形成的二级结构（$ {{att}} {{{C}} _ {{\ rm {bs}}}} $）和顶部（$ {{att}} {{{C}} _ {{\ rm {ts}}}} $）范例attCaadA7网站。我们发现两个序列都有两个结构 - 一个直的，规范的发夹和一个扭结的发夹。值得注意的是，重组首选$ {{att}} {{{C}} _ {{\ rm {bs}}}} $主要形成直发夹，而$ {{att}} {{{C}} _ {{\ rm {ts}}}} $优先采用扭结结构，只暴露一个完整的重组酶结合框。通过突变分析，我们在未配对的中间间隔区中鉴定了三个碱基，这可以反转优选的构象并增加$ {{att}} {{{C}} _ {{\ rm {ts}}}的重组频率。在体内。生物信息学筛选显示结构偏向于许多抗生素抗性盒attC位点的底链中的直的，规范的发夹构象。因此，我们预计结构微调可能是许多生物活性DNA发夹中的一种机制。

A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded single-stranded DNA hairpins-so-called attC sites-with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete. Here, we used high-resolution single-molecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom (${{att}}{{{C}}_{{\rm{bs}}}}$) and top (${{att}}{{{C}}_{{\rm{ts}}}}$) strands of the paradigmatic attCaadA7 site. We found for both sequences two structures-a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred ${{att}}{{{C}}_{{\rm{bs}}}}$ predominantly formed the straight hairpin, while the ${{att}}{{{C}}_{{\rm{ts}}}}$ preferentially adopted the kinked structure, which exposes only one complete recombinase binding box. By a mutational analysis, we identified three bases in the unpaired central spacer, which could invert the preferred conformations and increase the recombination frequency of the ${{att}}{{{C}}_{{\rm{ts}}}}$in vivo. A bioinformatics screen revealed structural bias toward a straight, canonical hairpin conformation in the bottom strand of many antibiotic resistance cassettes attC sites. Thus, we anticipate that structural fine tuning could be a mechanism in many biologically active DNA hairpins.

https://www.ncbi.nlm.nih.gov/pubmed/30566629