Chemical and Biochemical Studies on Pathological Aspect of Nitric Oxide

内源性一氧化氮是许多生理功能所必需的，有时在炎症中过量产生，因此致癌，但没有明确的证据报道。在这项研究中，我们获得了回答这个基本问题的重要数据。草氨酸（Oxa）是由no或hno2诱导的亚硝化脱氨作用产生的受损碱基，被认为是一种强诱变损伤。为了探究Oxa更详细的性质，本研究开发了含Oxa的DNA低聚物（Oxa- odn）的大规模制备：通过h2o2 -亚硝化从dGuo中得到Oxa的2'-脱氧核苷（脱氧oxanosine， dOxo），进行5'-0选择性三甲基化（DMT-dOxo收率70%）。随后，DMT-dOxo经常规磷酸化转化为DMT-dOxo-酰胺（收率，72.5%）。用该酰胺合成Oxa- odns， Oxa掺入偶联率和总合成率分别超过93%和85%。以含有dOxo或不含dOxo的DNA低聚物为模板和引物，研究了DNA聚合酶链延伸率，发现在Oxa的相反位点，T和C几乎相同，表明Oxa具有诱变性。我们对碱基切除修复（BER）系统进行了筛选，发现没有Oxa切除的酶活性，并发现Oxa-多胺加合物被核苷酸切除和重组修复系统修复，表明细胞Oxa形成，其他dna相关酶如连接酶和限制性内切酶将Oxa识别为gua。我们还发现，识别Oxa的BER酶与DNA双链中的Oxa交联是不可逆的，这种活性被加热消除，而非特异性结合DNA的组蛋白也与DNA双链中的Oxa交联，尽管速度缓慢。这种独特的交联将有助于捕获oxa识别和修复酶。目前正在对DNA中Oxa周围的结构进行核磁共振分析，以揭示Oxa这种独特性质的机制。少

Endogenous NO, necessary for many physiological functions, is sometimes overproduced in inflammation and therefore carcinogenic, however no clear evidence has been reported. In this study, we obtained important data to answer this essential question.1. Oxanine (Oxa), a damaged base generated from Gua by NO-or HNO2-induced nitrosative deamination, has been considered as a mutagen-potent lesion. In this study, for exploring more detailed properties of Oxa, large-scale preparation of Oxa-containing DNA oligomers (Oxa-ODN) was developed: 2'-deoxynucleoside of Oxa (deoxyoxanosine, dOxo) obtained from dGuo by HNO2-nitrosation was subjected to 5'-0-selective tritylation (yield of DMT-dOxo, 70%). Subsequently DMT-dOxo was converted by conventional phosphoramidation to DMT-dOxo-amidite (yield, 72.5%). The amidite was used for synthesizing Oxa-ODNs: The coupling yields for Oxa incorporation and the total synthesis of 25mer DNA were over 93 and 85 %, respectively.2. Using DNA oligomers with or wi … More thout dOxo as templates and primers, DNA polymerase chain elongation was investigated and we found that in the opposite site to Oxa, T and C are incorporated almost equally, indicating that Oxa is mutagenic.3. We performed screening for the base-excision repair (BER) system to find no enzymic activity for the excision of Oxa, and revealed that Oxa-polyamine adduct is repaired by the nucleotide excision and recombination repair system, indicative of the cellular Oxa formation and that other DNA-relevant enzymes such as ligase and restriction enzymes recognize Oxa as Gua.4. We also found that Oxa-recognizing BER enzymes cross-link with Oxa in the DNA duplex irreversibly and this activity is eliminated by heat, while histones binding DNA nonspecifically also crosslink Oxa in DNA duplexes although slowly. This unique crosslinking will be helpful for fishing Oxa-recognizing and-repairing enzymes.5. NMR analysis for the structure around Oxa in DNA is now being conducted to reveal the mechanism for such unique properties of Oxa. Less