Interactions Between Oligodeoxyribonucleotides and Their Utilizing Enzymes

1)限制性内切酶如何识别它们的特定序列是一个非常有趣的课题。为了阐明这个问题，人们已经制备了含有修饰碱基的DNA和寡核苷酸，并用它们来表征一些限制性内切酶的作用方式。我们以前报道过，在BGL II、MBO I和Sau 3Ai的识别序列中，含有7-去氮腺嘌呤、3-去氮腺嘌呤或N^6-甲基腺嘌呤的十脱氧核苷酸具有高度的耐水解性。然而，这些酶是否也与胸腺嘧啶相互作用并识别胸腺嘧啶功能尚不清楚。合成了在限制性内切酶BglⅡ、Sau3Ai、MBo I识别序列中含有尿嘧啶、5-溴、5-氰尿嘧啶和5-乙基尿嘧啶的十脱氧核苷酸。用5-溴胸腺嘧啶代替胸腺嘧啶的十核苷酸对所有限制性内切酶的敏感性基本相同。然而，含有尿嘧啶的十核苷酸对…具有很强的抗药性还有更多要攻击的。含有5-氰尿嘧啶的十核苷酸对Sau3Ai有很强的抵抗力，但能被Bgl II和Mbo I以及亲本十核苷酸所降解。含有5-乙基尿嘧啶的十核苷酸对Sau3Ai有很强的抵抗力，但对BglⅡ和MBO I有部分抵抗力。由此可见，5-取代基的大小是它们与酶相互作用的重要因素。3)合成了胞嘧啶-DNA甲基酶(HHA I甲基酶)的特异性抑制剂。4)研究了DNA聚合酶Klenow片段(E.Coli)对含有致突变碱基的寡核苷酸及其脱氧核糖苷5‘-三磷酸的识别作用。用Klenow片段将2‘-脱氧-N^6-甲氧基腺苷5’-三磷酸取代dGTP，用2-氨基-6-甲氧基氨基嘌呤2‘-脱氧核糖苷5’-磷酸取代dATP和dGTP。而含有N^6-甲氧基腺嘌呤或2-氨基-6-甲氧基氨基嘌呤的模板被Klenow片段识别为A和G。较少

1) How restriction endonucleases recognize their specific sequence is a subject of great interest. To elucidate this problem, DNAs and oligonucleotides containing modified bases have been prepared, and have been used to characterize the mode of action of some restriction enzymes.We have previously reported that decadeoxyribonucleotides containing 7-deazaadenine, 3-deazaadenine or N^6 -methyladenine in place of adenine in the recognition sequence of Bgl II, Mbo I and Sau 3AI were highly resistant to hydrolysis. However whether these enzymes also interact with and recognize the thymine function is not known. Decadeoxyribonucleotides containing uracil, 5-bromouracil, 5-cyanouracil and 5-ethyluracil in recognition sequences of restriction endonucleases Bgl II, Sau 3AI, Mbo I were synthesized. Decanucleotides containing 5-bromouracil in place of thymine had essentially the same susceptibility to all the restriction endonucleases. Uracil-containing decanucleotides were however very resistant … More to attack. Decanucleotides containing 5-cyanouracil were strongly resistant to hydrolysis by Sau 3AI, but were hydrolyzed by Bgl II and Mbo I as well as the parent decanucleotide. Decanucleotides containing 5-ethyluracil were strongly resistant to hydrolysis by Sau 3AI, but were partially resistant to hydrolysis by Bgl II and Mbo I. From these experiments, it is clear that the size of the 5-substituents is important factor when they interact with the enzyme.2) To find out the catalytic site of Eco RI, oligonucleotides containing 7-deazaadenine were synthesized. However these oligomers were found to be weak substrate of the anzyme.3) Specific inhibitors of cytosine-DNA methylase (Hha I methylase) was synthesized. Decanucleotides containing 5-fluorocytosine in recognition sequences of Hha I methylase were found to inhibit the enzyme activity.4) Recognition of oligonucleotides containing mutagenic nucleobases and their 5'-triphosphates of 2'-deoxyriboside by DNA polymerase Klenow fragment (E. Coli) was investigated. 2'-Deoxy-N^6 -methoxyadenosine 5'-triphosphate was incorporated into the primer instead of dGTP by Klenow fragment whereas 2-amino-6-methoxyaminopurine 2'-deozyriboside 5'-phosphate was incorporated in the place of dATP and dGTP. On the other hand, the template containing N^6 -methoxyadenine or 2-amino-6-methoxyaminopurine were recognized as both A and G by Klenow fragment. Less