Povirk L F, Goldberg I H
Proc Natl Acad Sci U S A. 1985 May;82(10):3182-6. doi: 10.1073/pnas.82.10.3182.
When defined-sequence DNA from the lacl region of plasmid pMC1 was treated with the nonprotein chromophore of neocarzinostatin in the presence of various thiols, the predominant lesions were direct strand breaks, occurring primarily at thymine and adenine residues. In the presence of glutathione, however, alkali-dependent strand breaks, occurring at certain cytosine residues, were also detected but were virtually absent when other thiols were used. Chromophore-induced release of free cytosine base from [3H]cytosine-labeled DNA was 2- to 3-fold greater with glutathione than with the other thiols. These results suggest that the alkali-dependent strand break is some form of apyrimidinic site. These sites were substrates for endonuclease IV of Escherichia coli, although a 5-fold greater concentration of enzyme was required for their cleavage than was required for cleavage of apurinic sites in depurinated DNA. These sites were also less sensitive to E. coli endonuclease VI (exonuclease III) by a factor of at least 5 and less sensitive to E. coli endonuclease III by a factor of at least 10. These and other results suggest that these sites are chemically different from normal apurinic/apyrimidine sites. When chromophore-induced apyrimidinic sites were quantitated as alkali-dependent breaks at 11 specific sites in the lacl gene, a correlation was found between occurrences of these lesions and the reported frequencies of G-C to A X T transitions at the same sites. All occurrences of the trinucleotide sequence A-G-C, including the ochre 21 mutational hot spot, were particularly prominent sites. The selective formation of endonuclease-resistant apyrimidinic sites at specific cytosine residues may explain the high frequency of G X C to A X T transitions in the mutational spectrum of neocarzinostatin.
当质粒pMC1的lacl区域的特定序列DNA在各种硫醇存在的情况下用新制癌菌素的非蛋白质发色团处理时，主要损伤是直接链断裂，主要发生在胸腺嘧啶和腺嘌呤残基处。然而，在谷胱甘肽存在下，也检测到在某些胞嘧啶残基处发生的碱依赖性链断裂，但使用其他硫醇时几乎不存在这种情况。与其他硫醇相比，谷胱甘肽使发色团诱导的游离胞嘧啶碱基从[3H]胞嘧啶标记的DNA中释放的量高出2至3倍。这些结果表明，碱依赖性链断裂是某种形式的脱嘧啶位点。这些位点是大肠杆菌内切核酸酶IV的底物，尽管切割它们所需的酶浓度比切割去嘌呤DNA中的无嘌呤位点所需的酶浓度高5倍。这些位点对大肠杆菌内切核酸酶VI（外切核酸酶III）的敏感性也至少低5倍，对大肠杆菌内切核酸酶III的敏感性至少低10倍。这些以及其他结果表明，这些位点在化学性质上与正常的无嘌呤/脱嘧啶位点不同。当将发色团诱导的脱嘧啶位点定量为lacl基因中11个特定位点处的碱依赖性断裂时，发现这些损伤的发生与同一位置报道的G-C到A×T转换频率之间存在相关性。三核苷酸序列A-G-C的所有出现情况，包括赭石21突变热点，都是特别突出的位点。在特定胞嘧啶残基处选择性形成抗内切核酸酶的脱嘧啶位点可能解释了新制癌菌素突变谱中G×C到A×T转换的高频率。
