Belguise-Valladier P, Fuchs R P
UPR 9003: Cancérogenèse et Mutägenèse Moléculaire et Structurale CNRS, Ecole Supérieure de Biotechnologie de Strasbourg, France.
J Mol Biol. 1995 Jun 23;249(5):903-13. doi: 10.1006/jmbi.1995.0347.
The strong rat liver carcinogen, N-2-acetylaminofluorene, forms mainly two types of guanine adducts at the C-8 position, the acetylaminofluorene adduct (dGuo-C8-AAF) and the aminofluorene adduct (dGuo-C8-AF). We have constructed different oligonucleotides bearing a single AF lesion at each of the guanine residues of the NarI mutagenesis hot spot (G1G2CG3CC) and analysed the structural distortion induced by this DNA adduct according to the sequence context. At position G1 and G2, the deformation induced by the AF adduct is smaller than the deformation induced by the corresponding acetylated form of this adduct (i.e. the AAF adduct at the G1 and G2), whereas both AF and AAF adducts induce a similar structural change when bound to G3. Single-stranded oligonucleotides modified with AF adducts were used in primer extension replication assays using purified DNA polymerases (PolIII holoenzyme, Klenow fragment (exo+ and exo-), Sequenase 2.0) and the data compared to the AAF containing substrates. Translesion synthesis (complete bypass) is found with all tested polymerases when AF adducts are bound to G1 or G2 while little or no bypass is seen when the AF adduct is bound to G3. On the other hand, irrespective of its position within the NarI sequence, AAF adducts completely block DNA synthesis. The results described in this paper show that the sole knowledge of the chemical structure of an adduct neither determines uniquely the conformational change it induces at the DNA level nor its replication properties. Indeed, although AF adducts are in most cases non-distorting adducts and as a consequence non-replication blocking lesions (as exemplified by adducts at G1 or G2), some AF adducts (as at position G3) behave almost as AAF adducts in terms of the structural distortion induced and its replication blocking property. These findings stress the strong modulation by the local sequence context of the structural and biological consequences of a given adduct.
强大鼠肝脏致癌物N - 2 - 乙酰氨基芴主要在鸟嘌呤的C - 8位形成两种类型的加合物，即乙酰氨基芴加合物（dGuo - C8 - AAF）和氨基芴加合物（dGuo - C8 - AF）。我们构建了在NarI诱变热点（G1G2CG3CC）的每个鸟嘌呤残基处带有单个AF损伤的不同寡核苷酸，并根据序列背景分析了这种DNA加合物诱导的结构畸变。在G1和G2位置，AF加合物诱导的变形小于该加合物相应乙酰化形式（即G1和G2处的AAF加合物）诱导的变形，而当与G3结合时，AF和AAF加合物均诱导相似的结构变化。用AF加合物修饰的单链寡核苷酸用于引物延伸复制试验，使用纯化的DNA聚合酶（PolIII全酶、Klenow片段（外切酶+和外切酶-）、测序酶2.0），并将数据与含AAF的底物进行比较。当AF加合物与G1或G2结合时，所有测试的聚合酶都能进行跨损伤合成（完全绕过），而当AF加合物与G3结合时，几乎看不到或根本没有绕过。另一方面，无论AAF加合物在NarI序列中的位置如何，它都会完全阻断DNA合成。本文所述结果表明，仅了解加合物的化学结构既不能唯一确定其在DNA水平诱导的构象变化，也不能确定其复制特性。实际上，尽管AF加合物在大多数情况下是无畸变加合物，因此是非复制阻断损伤（如G1或G2处的加合物所示），但一些AF加合物（如在G3位置）在诱导的结构畸变及其复制阻断特性方面几乎与AAF加合物表现相同。这些发现强调了局部序列背景对给定加合物的结构和生物学后果的强烈调节作用。
