Nearest-Neighbor Effects Modulate loxP Spacer DNA Chemical Shifts and Guide Oligonucleotide Design for Nuclear Magnetic Resonance Studies.
Nearest-Neighbor Effects Modulate loxP Spacer DNA Chemical Shifts and Guide Oligonucleotide Design for Nuclear Magnetic Resonance Studies.
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最近邻效应调节loxP间隔区DNA化学位移和用于核磁共振研究的指导寡核苷酸设计。
DOI：
10.1021/acs.biochem.1c00571
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发表时间：
2022-01-18
期刊：
影响因子：
2.9
通讯作者：
Foster, Mark P.
中科院分区：
文献类型：
--
作者：
Wagner, Nicole;Foster, Mark P.
Cre - loxP基因编辑工具能够对DNA进行位点特异性编辑，且不会留下必须由易错的细胞过程修复的损伤。Cre通过组装四聚体整合体复合物并形成霍利迪连接中间体，使两个34bp的loxP DNA位点发生重组，这些位点具有一对回文重组酶结合元件，两侧是不对称的8bp间隔区。重组是通过成对的催化酪氨酸残基对相对链间隔区中的特定磷酸二酯键进行协同亲核攻击而进行的。尽管Cre不与DNA的不对称间隔区进行碱基特异性接触，但它对其中一条链的初始切割表现出偏好，这表明未接触的8bp间隔区的内在特性导致了这种偏好。此外，对于使loxP间隔区成为Cre合适靶点的结构和动态特征知之甚少。为了能够对间隔区进行核磁共振波谱研究，我们旨在鉴定34bp loxP位点的一个片段，该片段保留间隔区的结构特征，同时最大限度地减少在较长寡核苷酸中出现的光谱拥挤和谱线展宽现象。不同长度的间隔区寡核苷酸以及一个改变链切割顺序的突变体之间的序列特异性化学位移差异，揭示了近邻和次近邻效应如何主导间隔区所经历的化学环境。我们已经鉴定出一种16bp的寡核苷酸，它保留了间隔区的结构环境，为基于核磁共振的结构测定和动力学研究奠定了基础。
The Cre-loxP gene editing tool enables site-specific editing of DNA without leaving lesions that must be repaired by error-prone cellular processes. Cre recombines two 34-bp loxP DNA sites that feature a pair of palindromic recombinase binding elements flanking an asymmetric 8-bp spacer region, via assembly of a tetrameric intasome complex and formation of a Holliday junction intermediate. Recombination proceeds by coordinated nucleophilic attack by pairs of catalytic tyrosine residues on specific phosphodiester bonds in the spacer regions of opposing strands. Despite not making base-specific contacts to the asymmetric spacer region of the DNA, Cre exhibits a preference for initial cleavage on one of the strands, suggesting that intrinsic properties of the un-contacted 8-bp spacer region give rise to this preference. Furthermore, little is known about the structural and dynamic features of the loxP spacer that make it a suitable target for Cre. To enable NMR spectroscopic studies of the spacer, we have aimed to identify a fragment of the 34-bp loxP site that retains the structural features of the spacer while minimizing the spectral crowding and line-broadening seen in longer oligonucleotides. Sequence-specific chemical shift differences between spacer oligos of different lengths, and of a mutant that inverts strand cleavage order, reveal how both nearest-neighbor and next-nearest-neighbor effects dominate the chemical environment experienced by the spacer. We have identified a 16-bp oligonucleotide that preserves the structural environment of the spacer, setting the stage for NMR-based structure determination and dynamics investigations.
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影响因子：
14.9
作者：
Sheren J;Langer SJ;Leinwand LA
通讯作者：
Leinwand LA
影响因子：
16.6
作者：
Bessen, Jeffrey L.;Afeyan, Lena K.;Liu, David R.
通讯作者：
Liu, David R.
影响因子：
5.6
作者：
Lee, L;Sadowski, PD
通讯作者：
Sadowski, PD
影响因子：
4.8
作者：
Kotar, Anita;Foley, Hannah N.;Keane, Sarah C.
通讯作者：
Keane, Sarah C.
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC