Asymmetric recognition of psoralen interstrand crosslinks by the nucleotide excision repair and the error-prone repair pathways
Asymmetric recognition of psoralen interstrand crosslinks by the nucleotide excision repair and the error-prone repair pathways
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DOI：
10.1006/jmbi.1999.2550
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发表时间：
1999-03-12
影响因子：
5.6
通讯作者：
Harel-Bellan, A
中科院分区：
文献类型：
--
作者：
Barre, FX;Asseline, U;Harel-Bellan, A
补骨脂素是一种具有呋喃和吡喃酮侧链的不对称光反应插层剂。当补骨脂素插入5‘-TPA-3’位时，在UVA照射下，补骨脂素可以与两条链上的THY-9残基反应，引入链间交联剂。利用补骨脂素偶联的三螺旋形成的寡核苷酸，补骨脂素链间交联物可以定点引入到URA3的编码序列中，URA3是酵母载体上携带的营养缺陷型标记。此外，通过三螺旋形成的寡核苷酸引入的交联链与补骨脂素的呋喃侧定向，与靶序列的特定链相关联。在此，我们检测了定位定位和定向定位的交联物在酵母细胞中的转化效率、突变频率和突变谱。我们发现，靶向突变的性质取决于交联链的取向：绕过吡喃酮加成的胸腺嘧啶产生T-->；A或T-->；C取代和A插入，而绕过呋喃加成的胸腺嘧啶产生T-->；G取代和G插入。因此，损伤的结构强烈影响跨损伤合成过程中加入的核苷酸的选择。此外，观察到的突变模式表明与转录的偶联，类似于在哺乳动物细胞中观察到的那样。最后，当补骨脂素交联物的吡喃酮连接在编码链上时，替换只影响编码链，而当吡喃酮连接在转录的链上时，它们影响两个链，这表明在细菌中纯化的uvrABC蛋白中观察到的补骨脂素交联物的切割偏好在活的真核细胞中是保守的。(C)1999年学术出版社。
Psoralen is an asymmetric photoreactive intercalator with a furane and a pyrone side. When intercalated at 5'-TpA-3' sites and upon UVA irradiation, the psoralen can react with the thy-nine residues on both strands, introducing an interstrand crosslink. Using psoralen-coupled triple-helix-forming oligonucleotides, psoralen interstrand crosslinks can be site-specifically introduced in the coding sequence of URA3, a yeast auxotrophic marker carried on plasmid vectors. Ln addition, crosslinks introduced via a triple-helix-forming oligonuleotide are oriented with the furane side of the psoralen associated with a specific strand of the target sequence. Here, the transformation efficiency, the mutation frequency and the mutational spectra of site-specifically placed and oriented cross-links were examined in yeast cells. We found that the nature of the targeted mutations depended on the crosslink orientation: bypass of the pyrone-adducted thymine yielded T --> A or T --> C substitutions and A insertions, while bypass of the furane-adducted thymine yielded T --> G substitutions and G insertions. Thus, the structure of the damage strongly influences the choice of the nucleotide incorporated during translesion synthesis. Ln addition, the observed pattern of mutagenesis suggests a coupling to transcription, similar to the one observed in mammalian cells. Finally, the substitutions affected only the coding strand when the pyrone link of the psoralen crosslink was on this strand, whereas they affected both strands when the pyrone link was on the transcribed strand, suggesting that the incision preference of psoralen crosslinks, which has been observed with purified uvrABC proteins in bacteria, is conserved in live eucaryotic cells. (C) 1999 Academic Press.