Iwaniec L M, Kroll J J, Roethel W M, Maybaum J
Department of Pharmacology, Upjohn Center for Clinical Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0504.
Mol Pharmacol. 1991 Mar;39(3):299-306.
Incorporation of the antileukemic agent 6-thioguanine (TG) into cellular DNA has been demonstrated to be a major determinant of its cytotoxicity. We have previously shown that complete replacement of G by TG within one DNA strand of the SV40 origin of replication can completely inhibit sequence-specific binding of the viral replication protein T antigen. The aim of the present study was to determine the effect of more selective TG substitutions on DNA-protein interactions, by utilizing the simpler base recognition sequence motifs of restriction endonucleases. In the first part of our study, we replaced G with TG in one or two of four possible sites within the duplex hexameric recognition sequence of BamHI (5'-G decreases GATCC-3'), by enzymatic extension of primed oligonucleotides. This extension was stalled, but not completely inhibited, at locations where insertion of consecutive TG moieties was required. Both strands of molecules containing a single substitution were cleaved by BamHI at reduced rates, with the substituted strand inhibited to a greater degree. In molecules containing two substitutions, neither strand was cut by BamHI. In contrast, we found that scission of these same mono- and disubstituted substrates by the less stringent isoschizomer MboI (5'-N decreases GATCN-3') was inhibited only slightly. In the second part of our study, we investigated the effect of analog substitution on scission by the type II-S enzymes AlwI and FokI, in order to separately determine the effects of restriction site modification versus scission site modification. We found that the reactivity of these enzymes was completely abolished by TG substitution within the recognition site, whereas substitution at the scission site had no effect. Our results demonstrate that infrequent TG substitutions within symmetric DNA sequences can inhibit sequence-specific interactions in an asymmetric fashion. In addition, although previous reports have shown that TG forms a relatively weak base pair with cytosine, it appears that the inhibition of restriction endonuclease-mediated cleavage resulting from TG incorporation is a function of the sequence requirements of the protein and not a general consequence of disrupted base-pairing at the recognition locus. These data support the idea that the cytotoxic consequences of TG incorporation may be due to inhibition of sequence-specific protein-DNA interactions.
抗白血病药物6-硫鸟嘌呤（TG）掺入细胞DNA已被证明是其细胞毒性的主要决定因素。我们之前已经表明，在SV40复制起点的一条DNA链中用TG完全取代G可完全抑制病毒复制蛋白T抗原的序列特异性结合。本研究的目的是通过利用限制性内切酶更简单的碱基识别序列基序来确定更具选择性的TG取代对DNA-蛋白质相互作用的影响。在我们研究的第一部分，我们通过引物寡核苷酸的酶促延伸，在BamHI（5'-G↓GATCC-3'）双链六聚体识别序列的四个可能位点中的一个或两个位点用TG取代G。在需要插入连续TG部分的位置，这种延伸受阻，但未被完全抑制。含有单个取代的分子的两条链都被BamHI以降低的速率切割，其中被取代的链受到更大程度的抑制。在含有两个取代的分子中，两条链都未被BamHI切割。相比之下，我们发现，不太严格的同裂酶MboI（5'-N↓GATCN-3'）对这些相同的单取代和双取代底物的切割仅略有抑制。在我们研究的第二部分，我们研究了类似物取代对II-S型酶AlwI和FokI切割的影响，以便分别确定限制性位点修饰与切割位点修饰的影响。我们发现，识别位点内的TG取代完全消除了这些酶的反应性，而切割位点的取代则没有影响。我们的结果表明，对称DNA序列中不常见的TG取代可以以不对称方式抑制序列特异性相互作用。此外，尽管先前的报告表明TG与胞嘧啶形成相对较弱的碱基对，但似乎TG掺入导致的限制性内切酶介导的切割抑制是蛋白质序列要求的函数，而不是识别位点碱基配对破坏的普遍结果。这些数据支持这样的观点，即TG掺入的细胞毒性后果可能是由于序列特异性蛋白质-DNA相互作用的抑制。
