AP sites are not significantly involved in mutagenesis by the (+)-anti diol epoxide of benzo[a]pyrene: the complexity of its mutagenic specificity is likely to arise from adduct conformational polymorphism.
AP sites are not significantly involved in mutagenesis by the (+)-anti diol epoxide of benzo[a]pyrene: the complexity of its mutagenic specificity is likely to arise from adduct conformational polymorphism.
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AP位点并不显着参与苯并[a]芘的( )-抗二醇环氧化物的诱变：其诱变特异性的复杂性可能是由加合物构象多态性引起的。
DOI：
10.1021/bi00077a009
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Loechler,EL
中科院分区：
文献类型：
--
作者：
Drouin,EE;Loechler,EL
1993年4月5日摘要：在以前的工作中，在大肠杆菌质粒pUB 3的supF基因中对苯并[a]芘[（+）-anfi-B [a] PDE]的（+）-抗二醇环氧化物诱导的突变进行了评分[Rodriguez & Loechler（1993）Biochemistry 32，1759]。pUB 3与（+）-抗-B [a] PDE反应，然后（1）立即转化到E.转化前在80 ℃下加热10 min。加热仅释放了一小部分加合物（~ 5%），对supF中大多数位点的致突变模式没有显著影响。然而，在主要的碱基替换热点处，在加热之前获得Gns，主要是G-T突变（87%），而在加热之后，G-T突变减少（45%），G-A（21%）和G-C（33%）突变变得更普遍。该结果的一个模型是，在加热之前，在Gns处的热不稳定加合物引起一种诱变模式，但在加热之后，不稳定加合物水解至脱嘌呤位点（AP位点），这引起第二种突变模式。为了检验这一点，研究了在80 ℃下加热10分钟从不稳定加合物产生的AP位点的作用。结果表明，当质粒pUB 3含有22（+）-α fi-B [a] PDE加合物时，0.6%（或更少）转化为AP位点，如在基于AP-内切核酸酶作用的测定中所测定的。在不涉及（+）-抗B [a] PDE加合物的单独研究中，估计了每个AP位点的突变频率（MF）。(In在这些实验中，通过在70 ℃/pH5.0下加热水解嘌呤的经典程序将AP位点引入pUB 3。事实上，基于三个标准，包括约75%的突变是GC-AT，通过该程序诱导的大多数突变可能是通过胞嘧啶脱氨基变为尿嘧啶而不是通过AP位点产生的。）根据（+）-抗-B [a] PDE加合物形成的AP位点的数目和MF/AP位点的估计，我们得出结论，当加合物质粒在转化前于80 ℃加热10分钟时，（+）-抗-B [a] PDE在supF中诱导的115个碱基置换突变中<2%可归因于AP位点。(With未加热的加合质粒，该限度为<~ 1%）。这使得AP位点不太可能显著地参与（+）-α « fi-B [a] PDE诱变，包括在Gns处，其中发现29%的碱基取代突变体。我们已经研究了苯并[a]芘（B [a] P ′）的诱变机制（Benasutti等人，1988; Loechler，1989; Loechler等人，1990; Mackay等人，1992; Rodriguez埃塔尔，1992; Rodriguez & Loechler，1993 a，B; Loechler，1993），一种众所周知的多环芳烃，被认为是一种庞大的诱变剂/致癌物。B [a] P可通过多种途径代谢[例如，菲利普斯等人，（1985），Marnett（1987），Cavalieri et al.（1990）和Devanesan et al.（1992）]，但其相应的（+）-抗二醇环氧化物[（+）-抗-B [a] PDE;图1]通常被认为是最重要的致癌衍生物（Conney，1982;菲利普斯，1983; Singer & Grunberger，1983; Harvey，1991），并且主要的DNA加合物是（+）-抗-B [a] P-N2-Gua（Cheng等人，1989; Sayer等人，1991年）。
Revised Manuscript Received April 5, 1993 abstract: In previous work, mutations induced by the (+)-anti diol epoxide of benzo [a] pyrene [(+)-anfi-B [a] PDE] were scored in the supF gene of the Escherichia coli plasmid pUB3 [Rodriguez & Loechler (1993) Biochemistry 32, 1759]. pUB3 was reacted with (+)-anti-B [a] PDE and then either (1) transformed immediately into E. coli or (2) heated at 80 C for 10 min prior to transformation. Heating only released a small fraction of adducts (~ 5%) and did not significantly affect the mutagenic pattern at most sites in supF. However, at the major base substitution hotspot, Gns, principally G—T mutations (87%) were obtained prior to heating, while after heating, G—T mutations decreased (45%) and G—A (21%) and G—C (33%) mutations became more prevalent. One model for this result is that prior to heating a heat-labile adduct at Gns causes one pattern of mutagenesis, but after heating the labile adduct is hydrolyzed to an apurinic site (AP site), which causes a second mutational pattern. To test this, a role for AP sites generated from labile adducts by heating at 80 C for 10 min is investigated. It is shown that when plasmid pUB3 contains 22 (+)-a «fi-B [a] PDE adducts, 0.6%(or fewer) are converted toAP sites as determined in an assay based uponthe action of an AP-endonuclease. In a separate line of investigation not involving (+)-anti-B [a] PDE adducts, mutation frequency (MF) per AP site is estimated.(In these experiments, AP sites were introduced into pUB3 by theclassic procedure of heating at 70 C/pH 5.0 to hydrolyze purines. In fact, the majority of mutants induced by this procedure probably arose via cytosine deamination to uracil and not via AP sites, based upon three criteria, including that~ 75% of the mutations were GC—AT.) Given the number of AP sites formed from (+)-anti-B [a] PDE adducts and the estimate of MF/AP site, we conclude that<~ 2% of the 115 base substitution mutations induced in supF by (+)-anfi-B [a] PDE can be attributed to AP sites when theadducted plasmid was heated at 80 C for 10 min prior to transformation.(With the unheated adducted plasmid, this limit is<~ 1%). This makes it unlikely that AP sites are significantly involved in (+)-a «fi-B [a] PDE mutagenesis, including at Gns, where 29% of base substitution mutants were found. The most likely alternative model for the heat-induced changes in mutational pattern at Gns, as well as the complexity of the mutagenic spectra of (+)-a «fi-B [a] PDE in general, is that a single adduct can adopt multiple conformations, each of which can cause different kinds of mutations, and factors such as heating and DNA sequence context can influence adduct conformation.We have been studying mechanisms of mutagenesis by benzo [a] pyrene (B [a] P)'(Benasutti et al., 1988; Loechler, 1989; Loechler et al., 1990; Mackay et al., 1992; Rodriguez etal., 1992; Rodriguez & Loechler, 1993a, b; Loechler, 1993), a well-known polycyclic aromatic hydrocarbon that is con-sidered a bulky mutagen/carcinogen. B [a] P may be me-tabolized by a variety of pathways [eg, Phillips et al.(1985), Marnett (1987), Cavalieri et al.(1990), and Devanesan et al.(1992)], but its corresponding (+)-anti diol epoxide [(+)-anti-B [a] PDE; Figure 1] is generally regarding to be the most important carcinogenic derivative (Conney, 1982; Phillips, 1983; Singer & Grunberger, 1983; Harvey, 1991) and the major DNA adduct is (+)-anfi-B [a] P-N2-Gua (Cheng et al., 1989; Sayer et al., 1991).
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