Activation-induced cytidine deaminase can target multiple topologies of double-stranded DNA in a transcription-independent manner
Activation-induced cytidine deaminase can target multiple topologies of double-stranded DNA in a transcription-independent manner
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DOI：
10.1096/fj.201903036rr
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发表时间：
2020-05-21
期刊：
影响因子：
4.8
通讯作者：
Larijani，Mani
中科院分区：
文献类型：
--
作者：
Branton，Sarah A.;Ghorbani，Atefeh;Larijani，Mani
激活诱导型胞苷脱氨酶(AID)突变免疫球蛋白基因，并在全基因组范围内发挥作用。AID靶向强健转录的基因，纯化的AID作用于单链(Ss)而不是双链(Ds)DNA寡核苷酸。因此，转录被认为是AID的单链DNA的生成者。以前研究转录和AID靶向之间的关系的无细胞研究采用了细菌集落计数试验，其中AID逆转了质粒底物中的抗生素耐药终止密码子，导致克隆形成。在这里，我们建立了一种新的分析方法，将不同拓扑结构的kb长的dsDNA与AID孵育，有或没有转录，然后直接测序。这种检测方法允许在没有转录或跨越一系列转录动力学的情况下，对AID靶向的突变频率和模式进行非选择性和深入的比较。我们发现，在没有转录的情况下，AID以超螺旋的形式靶向呼吸单链DNA，在较小的程度上，以松弛的dsDNA为靶标。最优转录仅以依赖于RNA聚合酶速度的方式适度增强了对超螺旋dsDNA的AID作用。这些数据表明，转录和AID靶向之间的相关性可能反映了导致AID呼吸的单链DNA斑块自然出现在脱色dsDNA中的转录，以及由于转录直接产生单链DNA。
Activation‐induced cytidine deaminase (AID) mutates immunoglobulin genes and acts genome‐wide. AID targets robustly transcribed genes, and purified AID acts on single‐stranded (ss) but not double‐stranded (ds) DNA oligonucleotides. Thus, it is believed that transcription is the generator of ssDNA for AID. Previous cell‐free studies examining the relationship between transcription and AID targeting have employed a bacterial colony count assay wherein AID reverts an antibiotic resistance stop codon in plasmid substrates, leading to colony formation. Here, we established a novel assay where kb‐long dsDNA of varying topologies is incubated with AID, with or without transcription, followed by direct sequencing. This assay allows for an unselected and in‐depth comparison of mutation frequency and pattern of AID targeting in the absence of transcription or across a range of transcription dynamics. We found that without transcription, AID targets breathing ssDNA in supercoiled and, to a lesser extent, in relaxed dsDNA. The most optimal transcription only modestly enhanced AID action on supercoiled dsDNA in a manner dependent on RNA polymerase speed. These data suggest that the correlation between transcription and AID targeting may reflect transcription leading to AID‐accessible breathing ssDNA patches naturally occurring in de‐chromatinized dsDNA, as much as being due to transcription directly generating ssDNA.