BLEOMYCIN STRUCTURE & FUNCTION: NMR SPECTROSCOPY

博莱霉素(BLM)是一类抗肿瘤抗生素 目前临床上用于治疗睾丸癌，头部 以及宫颈癌和霍奇金氏病。建立 细胞内主要靶点(S)及其作用机制(S) 因此，博莱曼的细胞毒性和治疗效果非常显著 利息。BLM已经知道一段时间了，它可以同时影响两个单一的 DNA在体外和体内的链(Ss)和双链(Ds)断裂。 这些DS中断被认为是主要的促成因素 在博莱姆的细胞毒性中。一个关于单个分子如何 可以在不解离的情况下对两条DNA进行切割 根据CM[R]最近获得的结构数据提出了建议。 在这个模型中，博莱姆分子的双噻唑单元被认为是 经历重新定位金属结合区的反式到顺式翻转 在第二链上的分子。铁的过氧化氢形式 BLM(激活的BLM)已被证明在机械研究中抽象 DNA核糖部分的4‘-H。一旦去掉了4‘-H， 形成了两个主要的DNA损伤。磷酸乙醇酸损伤可以是 以依赖氧的方式形成，并且4‘-酮基基位可以是 由氧不依赖的途径形成的。的结构。 磷酸乙醇酸损伤可能是了解DS的关键 BLM对DNA的切割。在此之前，一种磷酸乙酸酯损伤 含有GTAC序列的序列已在本实验室合成。这个 选择GTAC序列是因为它是DS切割的热点 DS：SS裂解比I：3.获得好数据的关键 因为这项工作是将寡核苷酸合成为双链 发夹与乙二醇间隔物相连。质子 化学位移任务已经完成，2D的建模 在750兆赫核磁共振上收集的核磁共振数据目前正在进行中。这个 寡核苷酸也用过氧化钴形式滴定。 BLM，它是活性铁BLM的拟议类似物。这 滴定将在附近用更浓缩的样品重复进行 未来。该样本的准备工作正在进行中。的综合体 钴-博莱曼和DNA可能是了解其发病机制的关键 DS卵裂。可能会将数据从 从完整的DNA和DNA中提取的数据 在联噻唑区检测到反式到顺式的异构化。另一个 含有磷酸乙醇酸损伤的寡核苷酸样本已经 也做好了准备。这种寡核苷酸也是一个双重发夹。 由六乙二醇间隔物连接。然而，这篇文章包含了 GGCC序列。有趣的是，这个序列不能经历DS 通过BLM分子进行切割。因此，该序列起到了 对照实验。该DNA的数据已在D20和 90%H20/10%D20。化学位移的赋值在 进步。这种寡核苷酸也将用钴滴定 过氧化氢BLm。如果这个寡核苷酸产生一对一的复合体 在BLM中，将这些数据与 来自GTAC文章的数据。4‘-酮基位的合成是 实验室里也在进行中。这种损伤是一种人工合成的挑战 到目前为止，制造这种损伤的努力还没有成功。然而， 已经对这个问题采取了一种新的方法。一个 4‘-叠氮-2’-脱氧尿苷部分已经合成，将被 利用聚合酶和一种酶结合到DNA中。后续 还原将产生4‘-酮基底位。从4‘-keto开始 基本部位目前不能用于结构测定，一个 近亲，一个4‘-羟基的基本位置正在用2D核磁共振研究。 同样，这些数据是在CMR的750 MHz仪器上获得的。这 在GTAC序列上下文中正在研究基本位置 与磷酸乙醇酸盐损伤进行比较。它的模型是 含有碱性位点的双链13-聚体处于 精致。有趣的是，这种基本的两种截然不同的构象 GTAC地区的遗址数量相等。这导致了 DNA修复酶识别这一损伤部位的问题。 可以合理地假设其中一种构象是 优先被DNA修复酶识别，如人类 脱嘌呤/脱嘧啶核酸内切酶(APEI)。总而言之，2D的建模 在750 MHz仪器上获得的核磁共振数据处于不同阶段 基本部位和磷酸乙醇酸病变的精细化。 2D核磁共振数据的分配正在进行中，用于其他 与钴-博莱姆结合和不结合的磷酸乙醇酸损伤。

The bleomycins (BLM's) are a family of antitumor antibiotics presently used clinically in the treatment of testicular cancer, head and neck carcinoma as well as Hodgkin's disease. Establishing the major intracellular target(s) and the mechanism(s) responsible for BLM's observed cytotoxicity and therapeutic efficacy is thus of great interest. BLM has been known for some time to effect both single strand (ss) and double strand (ds) breaks of DNA in vitro and in vivo. These ds breaks have been proposed to be the major contributing factor in BLM's cytotoxicity. A model for the way in which a single molecule of BLM can effect cleavage on two strands of DNA without dissociating has been proposed based on recent structural data acquired at CM[R. In this model, the bithiazole unit of the BLM molecule is thought to undergo a trans to cis flip that repositions the metal binding region of the molecule at the second strand. The hydroperoxide form of iron BLM (activated BLM) has been shown in mechanistic studies to abstract the 4'-H of the DNA ribose moiety. Once the 4'-H has been removed, two major DNA lesions are formed. A phosphoglycolate lesion can be formed in an oxygen dependent manner, and a 4'-keto abasic site can be formed by an oxygen independent pathway. The structure of the phosphoglycolate lesion may present the key to understanding the ds cleavage of the DNA by BLM. Previously, a phosphoglycolate lesion containing the GTAC sequence had been synthesized in our lab. The GTAC sequence was chosen since it is a hot spot for ds cleavage with a ratio of ds : ss cleavage of I : 3. The key to acquiring good data for this piece had been to synthesize the oligonucleotide as a double hairpin connected with hexaethyleneglycol spacers. The proton chemical shift assignments have been completed and modeling of the 2D NMR data collected on a 750 MHz NMR is currently underway. The oligonucleotide had also been titrated with a cobalt hydroperoxy form of BLM, which is a proposed analog of activated iron BLM. This titration will be repeated with a more concentrated sample in the near future. Preparation of this sample is underway. The complex of cobalt BLM and DNA may hold the key to understanding the mechanism of ds cleavage. It may be possible to cont rast the data from the phosphoglycolate lesion with the data from an intact piece of DNA and detect a trans to cis isomerization in the bithiazole region. Another oligonucleotide sample containing the phosphoglycolate lesion has been prepared as well. This oligonucleotide is also a double hairpin linked by hexaethyleneglycol spacers. However, this piece contains the GGCC sequence. Interestingly, this sequence cannot undergo ds cleavage via the BLM molecule. This sequence thus functions as a control experiment. Data for this DNA has been collected in D20 and 90%H20/10%D20. The assignments of the chemical shifts are in progress. This oligonucleotide will also be titrated with a cobalt hydroperoxy BLM. If this oligonucleotide yields a one to one complex with the BLM, it will be interesting to contrast this data with the data from the GTAC piece. The synthesis of the 4'-keto abasic site is also in progress in the lab. This lesion is a synthetic challenge and efforts to make this lesion have thus far been unsuccessful. However, a new approach to this problem has been taken. A 4'-azido-2'-deoxyuridine moiety has been synthesized that will be incorporated into DNA using a polymerase and a kinase. Subsequent reduction will yield the 4'-keto abasic site. Since the 4'-keto abasic site is currently unavailable for structure determination, a close relative, a 4'-OH abasic site is being studied by 2D NMR. Again, this data was acquired on the 750 MHz instrument at CMR. This abasic site is being studied in the GTAC sequence context for comparison with the phosphoglycolate lesion. The modeling of this duplex 13-mer containing the abasic site is in the final stages of refinement. Interestingly, two distinct conformations of this abasic site in the GTAC region are present in equal amounts. This leads to the question of recognition of this damage site by DNA repair enzymes. It is reasonable to postulate that one of these conformations is recognized preferentially by DNA repair enzymes such as human apurinic/apyrimidinic endonuclease (APEI). In summary, modeling of 2D NMR data acquired on the 750 MHz instrument are at various stages of refinement for the abasic site and the phosphoglycolate lesions. Assignments of the 2D NMR data are in progress for additional phosphoglycolate lesions with and without Co-BLM bound.