THYMINE RING SATURATION AND FRAGMENTATION PRODUCTS - LESION BYPASS, MISINSERTION AND IMPLICATIONS FOR MUTAGENESIS
THYMINE RING SATURATION AND FRAGMENTATION PRODUCTS - LESION BYPASS, MISINSERTION AND IMPLICATIONS FOR MUTAGENESIS
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DOI：
10.1016/0165-1218(93)90092-r
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发表时间：
1993-05-01
期刊：
影响因子：
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通讯作者：
WALLACE, S
中科院分区：
文献类型：
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作者：
EVANS, J;MACCABEE, M;WALLACE, S
我们使用胸腺嘧啶乙二醇和二氢胸腺嘧啶作为自由基与DNA嘧啶相互作用产生的典型环饱和产物，尿素糖苷和β -脲异丁酸（UBA）作为嘧啶环断裂产物的模型。我们已经证明胸腺嘧啶乙二醇和环断裂产物尿素和β -脲异丁酸，以及基本位点，是体外DNA聚合酶的强阻滞。相比之下，二氢胸腺嘧啶对测试的任何聚合酶都没有阻滞作用。对于胸腺嘧啶乙二醇，终止位点在假定的病变相反，而对于环碎片化产物，终止位点主要是在病变之前的一个碱基。这些和其他数据表明，胸腺嘧啶乙二醇编码A，并且一个碱基稳定地插入到损伤的对面，而当一个碱基插入到非编码病变的对面时，它被DNA聚合酶1的3 - bbb50外切酶活性除去。尽管胸腺嘧啶乙二醇、尿素和UBA具有阻断病变的作用，但在某些特定的序列背景下，它们可以低频绕过。当模型病变单独引入单链生物活性DNA时，我们发现胸腺嘧啶乙二醇、尿素、β -脲异丁酸和基位都是致命病变，激活效率为1，而二氢胸腺嘧啶则不是。因此，体外研究预测了体内结果。当生物活性单链DNA在紫外线诱导的大肠杆菌细胞中存活时，当复制阻滞被释放时，没有观察到含有尿素或基本位点的DNA存活的增加，这表明这些病变的低效旁路。相比之下，β -脲异丁酸的存活率略有提高，转染含有胸腺嘧啶二醇的DNA显着重新激活。当使用含有大肠杆菌靶基因的f1-K12杂交DNA测量独特病变诱导突变时，发现胸腺嘧啶二甘醇和二氢胸腺嘧啶作为致突变前病变是无效的，这表明它们在体内和体外主要编码a。相比之下，尿素和β -尿嘧啶异丁酸是有效的致突变前病变，β -尿嘧啶异丁酸比尿素苷有效约4-5倍。它们的诱变率与嘌呤的碱基位点大致相同。对这些环片段化产物产生的突变的序列分析表明，产生的突变既依赖于病变，也依赖于序列上下文。本文讨论了旁路效率和病变导向误插入在诱变中可能起的作用。
We have used thymine glycol and dihydrothymine as representative ring saturation products resulting from free-radical interaction with DNA pyrimidines, and urea glycosides and beta-ureidoisobutyric acid (UBA) as models for pyrimidine-ring fragmentation products. We have shown that thymine glycol and the ring-fragmentation products urea and beta-ureidoisobutyric acid, as well as abasic sites, are strong blocks to DNA polymerases in vitro. In contrast, dihydrothymine is not a block to any of the polymerases tested. For thymine glycol, termination sites were observed opposite the putative lesions, whereas for the ring-fragmentation products, the termination sites were primarily one base prior to the lesion. These and other data have suggested that thymine glycol codes for an A, and that a base is stably inserted opposite the damage, whereas when a base is inserted opposite the non-coding lesions, it is removed by the 3 --> 5 exonuclease activity of DNA polymerase 1. Despite their efficiency as blocking lesions, thymine glycol, urea and UBA can be bypassed at low frequency in certain specific sequence contexts.When the model lesions were introduced individually into single-stranded biologically active DNA, we found that thymine glycol, urea, beta-ureidoisobutyric acid, and abasic sites were all lethal lesions having an activation efficiency of 1, whereas dihydrothymine was not. Thus the in vitro studies predicted the in vivo results. When the survival of biologically active single-stranded DNA was examined in UV-induced Escherichia coli cells where the block to replication was released, no increase in survival was observed for DNA containing urea or abasic sites, suggesting inefficient bypass of these lesions. In contrast, beta-ureidoisobutyric acid survival was slightly enhanced, and transfecting DNA containing thymine glycols was significantly reactivated. When mutation induction by unique lesions was measured using f1-K12 hybrid DNA containing an E. coli target gene, thymine glycols and dihydrothymine were found to be inefficient as premutagenic lesions, suggesting that in vivo, as in vitro, they primarily code for A. In contrast, urea and beta-ureidoisobutyric acid were efficient premutagenic lesions, with beta-ureidoisobutyric acid being about 4-5-fold more effective than urea glycosides, which have approximately the same rate of mutation induction as abasic sites from purines. Sequence analysis of the mutations resulting from these ring-fragmentation products shows that the mutations produced are both lesion and sequence context dependent. The possible roles that bypass efficiency and lesion-directed misinsertion might play in mutagenesis are discussed.