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首页> 外文期刊>Journal of Molecular Biology >Alkylating agent and chromatin structure determine sequencecontext-dependent formation of alkylpurines
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Alkylating agent and chromatin structure determine sequencecontext-dependent formation of alkylpurines

机译：烷基化剂和染色质结构决定了烷基嘌呤序列依赖上下文的形成

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We determined the adduct maps of S(N)1 and S(N)2 alkylating agents in cultured human cells (in vivo) and in vitro to probe DNA-protein interactions along sequences of the promoter and exon 1 of the Fragile-X mental retardation 1 (FMR1) gene. Using ligation-mediated polymerase chain reaction (LMPCR), we compared the piperidine-sensitive alkylpurines sites generated by treating cultured cells tin vivo) and naked DNA (in vitro) with S(N)1 (N-methyl-N-nitrosourea, N-nitroso(acetoxymethyl)methylamine and 1-methyl-3-nitro-1-nitrosoguanidine) and S(N)2 alkylating agents (dimethyl sulfate (DMS), methane sulfonic acid methyl ester, iodo methane, diethyl sulfate, methane sulfonic acid ethyl ester and iodo ethane). The FMR1 promoter has four sites where DNA-protein interactions are observed. In these regions, the S(N)1 methylating agent reactions produced only hypo-reactive sites. In contrast, iodoalkane S(N)2 alkylating agents (MeI and EtI) reactions generated only hyper-reactive sites. Although there are hyper-reactive sites for the other S(N)2 reagents, the hyper-reactive site at +14 on the FMR1 map is more pronounced for the sulfate and sulfonate-derived alkylating agents than for the iodoalkanes. However, DMS modification in the presence of methyl sulfone, a compound that does not alkylate DNA, eliminates the hyper-reactive site observed at +14. This suggests that the electron-rich oxygen atoms of the sulfate and sulfonate-derived S(N)2 alkylating agent structure position the alkylating moiety to the neighboring N-7-guanine position to favor alkyl transfer to the guanine. Using KMnO4 to probe for single-strand DNA, an unpaired cytosine base was detected at the 5'-side of the hyperreactive guanine base at position +14, consistent with the formation of a local DNA single-strand bulge. Ln conclusion, we show that the sequence context-dependent formation of alkylpurines is determined by the chemical nature of the alkylating agent, the DNA sequence context, chromatin structure, and the presence of other non-reactive molecules that can inhibit alkylation.
机译：我们确定了S（N）1和S（N）2烷化剂在培养的人细胞中（体内）和体外的加合物图，以沿着Fragile-X mental的启动子和外显子1的序列探测DNA-蛋白质相互作用延迟1（FMR1）基因。使用连接介导的聚合酶链反应（LMPCR），我们比较了用S（N）1（N-甲基-N-亚硝基脲，N）处理通过体外培养培养细胞和体外裸DNA产生的哌啶敏感性烷基嘌呤位点。 -亚硝基（乙酰氧基甲基）甲胺和1-甲基-3-硝基-1-亚硝基胍和S（N）2烷基化剂（硫酸二甲酯（DMS），甲烷磺酸甲酯，碘甲烷，硫酸二乙酯，甲烷磺酸乙酯酯和碘乙烷）。 FMR1启动子有四个站点，在其中观察到DNA-蛋白质相互作用。在这些区域中，S（N）1甲基化剂反应仅产生低反应位。相反，碘代烷S（N）2烷基化剂（MeI和EtI）反应仅产生高反应位。尽管其他S（N）2试剂有高反应位，但硫酸盐和磺酸盐衍生的烷基化剂在FMR1图谱上+14处的高反应位比碘代烷烃更明显。但是，在甲基砜（一种不会使DNA烷基化的化合物）的存在下进行DMS修饰，可以消除在+14处观察到的高反应位点。这表明硫酸盐和磺酸盐衍生的S（N）2烷基化剂结构的富电子氧原子将烷基化部分定位在相邻的N-7-鸟嘌呤位置上，从而有利于烷基转移至鸟嘌呤。使用KMnO4探测单链DNA，在+14位高反应性鸟嘌呤碱基的5'端检测到未配对的胞嘧啶碱基，这与局部DNA单链凸起形成一致。总之，我们表明烷基嘌呤的序列背景依赖性形成取决于烷基化剂的化学性质，DNA序列背景，染色质结构以及其他可抑制烷基化的非反应性分子的存在。

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