BLEOMYCIN-INDUCED DNA LESIONS AT MUTATIONAL HOT-SPOTS - IMPLICATIONS FOR THE MECHANISM OF DOUBLE-STRAND CLEAVAGE
BLEOMYCIN-INDUCED DNA LESIONS AT MUTATIONAL HOT-SPOTS - IMPLICATIONS FOR THE MECHANISM OF DOUBLE-STRAND CLEAVAGE
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DOI：
10.1073/pnas.87.21.8350
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发表时间：
1990-11-01
影响因子：
11.1
通讯作者：
POVIRK, LF
中科院分区：
文献类型：
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作者：
STEIGHNER, RJ;POVIRK, LF
使用各种末端标记，确定序列的DNA底物，我们研究了博莱霉素诱导的损伤在几个G.cntdot.C碱基对，对应于突变热点。检测到的最常见的病变是单链断裂和C残基处的单个脱嘌呤/脱嘧啶（AP）位点，表明这是损伤的主要部位。链断裂和AP位点也发生在二次损伤位点，但频率较低。互补链中直接相对的G残基。 然而，在二级网站的损害发生时，只有一个链断裂存在于初级网站，和AP网站在初级网站从来没有伴随着密切反对的损害互补链。因此，在原发性损伤部位形成链断裂是继发性损伤部位发生攻击的必要条件，但不是充分条件。类似的模式在其他序列攻击博莱霉素，虽然初级和二级网站有时交错的一个核苷酸的位置，而不是直接相反。这些和其他结果表明了双链切割的机制，其中博来霉素在第一链断裂形成期间被再活化，并且再活化的药物随后在通常不是博来霉素诱导的切割位点的特定位置攻击互补链。活化的博来霉素的再生可由Fe（III）·博来霉素和脱氧核糖的4 ″-过氧衍生物之间的反应产生，两者都是在链断裂形成期间产生的。
Using various end-labeled, defined-sequence DNA substrates, we examined bleomycin-induced damage at several G.cntdot.C base pairs which correspond to mutational hot spots. The most frequent lesions detected were single-strand breaks and single apurinic/apyrimidinic (AP) sites at the C residue, suggesting that this was the primary site of damage. Strand breaks and AP sites also occurred, but less frequently, at a secondary damage site.sbd.i.e., the directly opposed G residue in the complementary strand. However, damage at the secondary site occurred only when a strand break was present at the primary site, and AP sites at the primary site were never accompanied by closely opposed damage in the complementary strand. Thus, formation of a strand break at the primary damage site was a necessary though not sufficient condition for attack at the secondary site. Similar patterns were seen at other sequences attacked by bleomycin, although primary and secondary sites were sometimes staggered by one nucleotide position rather than directly opposed. These and other results suggest a mechanism of double-strand cleavage in which bleomycin is reactivated during formation of the first strand break, and the reactivated drug subsequently attacks the complementary strand at a specific position which is not normally a site of bleomycin-induced cleavage. Regeneration of activated bleomycin could result from a reaction between Fe(III).cntdot.bleomycin and a 4''-peroxyl derivative of deoxyribose, both produced during formation of the strand break.