On the formation and properties of interstrand DNA-DNA cross-links forged by reaction of an abasic site with the opposing guanine residue of 5'-CAp sequences in duplex DNA.
On the formation and properties of interstrand DNA-DNA cross-links forged by reaction of an abasic site with the opposing guanine residue of 5'-CAp sequences in duplex DNA.
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DOI：
10.1021/ja308119q
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发表时间：
2013-01-23
影响因子：
15
通讯作者：
Gates, Kent S.
中科院分区：
文献类型：
--
作者：
Johnson, Kevin M.;Price, Nathan E.;Wang, Jin;Fekry, Mostafa I.;Dutta, Sanjay;Seiner, Derrick R.;Wang, Yinsheng;Gates, Kent S.
我们最近报道，双链 DNA 中脱碱基 (Ap) 位点的醛基残基可以通过与相对链上的鸟嘌呤残基反应产生链间交联。这一发现很有趣，因为链间交联的高度有害性质表明，即使是少量的Ap衍生的交联也可能对细胞DNA中Ap位点产生所产生的生物学后果做出重大贡献。在还原胺化（NaCNBH3，pH 5.2）条件下孵育含有中心 5'-CAp 序列的 21 bp 双链体，产生的交联 DNA 产量比之前报道的要高得多。在pH 7下，在没有还原剂的情况下，这些含Ap的双链体也产生交联双链体，其在变性聚丙烯酰胺凝胶上很容易检测到。交联形成对反应条件不高度敏感，并且一旦形成，交联对于各种后处理条件都是稳定的。多项实验（包括 MALDI-TOF 质谱、凝胶迁移率、Ap 醛的甲氧基胺封端、肌苷换鸟嘌呤置换、羟基自由基足迹和 LCMS/MS）的结果与涉及 Ap 醛残基与 5'-CAp 序列中相对鸟嘌呤残基的 N2-氨基发生可逆反应以生成半缩醛胺、亚胺或环状半缩醛胺交联的交联机制一致。 (7-10) 在还原胺化 (NaCNBH3/pH 5.2) 条件下不可逆地转化为稳定的胺键。一项实验进一步支持了环外 N2-氨基在此反应中的重要性，该实验表明，在不存在 NaCNBH3 的情况下，在交联位点安装 2-氨基嘌呤-胸腺嘧啶碱基对可在中性 pH 条件下产生高产率 (15-30%) 的交联双链体。
We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5′-CAp sequence under conditions of reductive amination (NaCNBH3, pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions and, once formed, the cross-link was stable to a variety of work-up conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LCMS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N2-amino group of the opposing guanine residue in 5′-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH3/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N2-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH3.
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