Mapping of cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) DNA adducts over >3000 nucleotides was carried out using a replication blockage assay. The sites of inhibition of modified T4 DNA polymerase, also referred to as stop sites, were analyzed to determine the effects of local sequence context on the distribution of intrastrand cisplatin cross-links. In a 3120 base fragment from replicative form M13mp18 DNA containing 24.6% guanine, 25.5% thymine, 26.9% adenine and 23.0% cytosine, 166 individual stop sites were observed at a bound platinum/nucleotide ratio of 1-2 per thousand. The majority of stop sites (90%) occurred at G(n>2) sequences and the remainder were located at sites containing an AG dinucleotide. For all of the GG sites present in the mapped sequences, including those with Gn(>)2, 89% blocked replication, whereas for the AG sites only 17% blocked replication. These blockage sites were independent of flanking nucleotides in a sequence of N(1)G*G*N(2) where N(1), N(2) = A, C, G, T and G*G* indicates a 1,2-intrastrand platinum cross-link. The absence of long-range sequence dependence was confirmed by monitoring the reaction of cisplatin with a plasmid containing an 800 bp insert of the human telomere repeat sequence (TTAGGG)(n). Platination reactions monitored at several formal platinum/nucleotide ratios or as a function of time reveal that the telomere insert was not preferentially damaged by cisplatin. Both replication blockage and telomere-insert plasmid platination experiments indicate that cisplatin 1,2-intrastrand adducts do not form preferentially at G-rich sequences in vitro.

译文

:使用复制阻滞测定法对> 3000个核苷酸的顺式-二甲基二氯铂(II)(顺式-DDP,顺铂)DNA加合物进行映射。分析了修饰的T4 DNA聚合酶的抑制位点,也称为终止位点,以确定局部序列背景对链内顺铂交联分布的影响。在含有14.6%鸟嘌呤,25.5%胸腺嘧啶,26.9%腺嘌呤和23.0%胞嘧啶的复制型M13mp18 DNA的3120个碱基片段中,观察到166个单独的终止位点,结合的铂/核苷酸比率为1-2 /千。大部分终止位点(90%)出现在G(n> 2)序列上,其余位点位于含有AG二核苷酸的位点上。对于映射序列中存在的所有GG位点,包括那些具有Gn(>)2的位点,有89%的位点阻止了复制,而对于AG位点,只有17%的位点阻止了复制。这些阻断位点独立于N(1)G * G * N(2)序列中的侧翼核苷酸,其中N(1),N(2)= A,C,G,T和G * G *表示1 ,2-链内铂交联。通过监测顺铂与含有人端粒重复序列(TTAGGG)(n)的800 bp插入片段的质粒的反应,可以确认不存在长程序列依赖性。在几个正式的铂/核苷酸比率或时间的函数下监测的铂化反应表明,端粒插入物不会被顺铂优先破坏。复制阻滞和端粒插入质粒电镀实验均表明,在体外富含G的序列中,顺铂1,2-内链加合物并非优先形成。

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