Ober Matthias, Müller Heiko, Pieck Carsten, Gierlich Johannes, Carell Thomas
Department of Chemistry and Biochemistry Ludwig-Maximilians-University Munich, D-81377 Munich, Germany.
J Am Chem Soc. 2005 Dec 28;127(51):18143-9. doi: 10.1021/ja0549188.
The 2,6-diamino-4-hydroxy-5-formamidopyrimidine of 2'-deoxyguanosine (FaPydG) is one of the major DNA lesions found after oxidative stress in cells. To clarify the base pairing and coding potential of this major DNA lesion with the aim to estimate its mutagenic effect, we prepared oligonucleotides containing a cyclopentane based analogue of the DNA lesion (cFaPydG). In addition, oligonucleotides containing the cyclopentane analogue of 2'-deoxyguanosine (cdG), and oligonucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were synthesized. The thermodynamic stability of duplexes containing these building blocks and all canonical counterbases were determined by concentration dependent melting-point measurements (van't Hoff plots). The data reveal that cFaPydG greatly destabilizes a DNA duplex (DeltaDeltaG degrees (298K) approximately 2-4 kcal mol(-1)). The optimal base pairing partner for the cFaPydG lesion is dC. Investigation of duplexes containing dG and cdG shows that the effect of substituting the deoxyribose by a cyclopentane moiety is marginal. The data also provide strong evidence that the FaPydG lesion is unable to form a base pair with dA. Our computational studies indicate that the syn-conformation required for base pairing with dA is energetically unfavorable. This is in contrast to 8-oxodG for which the syn-conformation represents the energetic minimum. Kinetic primer extension studies using S. cerevisiae Pol eta reveal that cFaPydG is replicated in an error-free fashion. dC is inserted 2-3 orders of magnitude more efficiently than dT or dA, showing that FaPydG is a lesion which retains the coding potential of dG. This is also in contrast to 8-oxodG, for which base pairing with dC and dA was established.
2'-脱氧鸟苷的2,6-二氨基-4-羟基-5-甲酰胺基嘧啶（FaPydG）是细胞氧化应激后发现的主要DNA损伤之一。为了阐明这种主要DNA损伤的碱基配对和编码潜力，以评估其诱变作用，我们制备了含有基于环戊烷的DNA损伤类似物（cFaPydG）的寡核苷酸。此外，还合成了含有2'-脱氧鸟苷环戊烷类似物（cdG）的寡核苷酸以及含有8-氧代-7,8-二氢-2'-脱氧鸟苷（8-oxodG）的寡核苷酸。通过浓度依赖性熔点测量（范特霍夫图）确定了含有这些结构单元和所有标准互补碱基的双链体的热力学稳定性。数据表明，cFaPydG极大地破坏了DNA双链体的稳定性（ΔΔG°(298K)约为2 - 4 kcal mol⁻¹）。cFaPydG损伤的最佳碱基配对伙伴是dC。对含有dG和cdG的双链体的研究表明，用环戊烷部分取代脱氧核糖的影响很小。数据还提供了有力证据，表明FaPydG损伤无法与dA形成碱基对。我们的计算研究表明，与dA碱基配对所需的顺式构象在能量上是不利的。这与8-oxodG相反，对于8-oxodG，顺式构象代表能量最小值。使用酿酒酵母Pol eta进行的动力学引物延伸研究表明，cFaPydG以无错误方式复制。dC的插入效率比dT或dA高2 - 3个数量级，表明FaPydG是一种保留了dG编码潜力的损伤。这也与8-oxodG相反，8-oxodG已确定与dC和dA碱基配对。
