INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

大分子拥挤对生化系统的影响

• 批准号: 3964707
• 负责人: S B ZIMMERMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3964707
• 关键词: DNA; DNA repair; bacterial DNA; bacteriophage T4; bacteriophage lambda; chemical binding; circular DNA; deoxyribonucleotides; enzyme substrate; ethylene glycols; glycogen; macromolecule; molecular biology; molecular genetics; oligonucleotides; polymers; virus DNA

T4 polynucleotide kinase rapidly loses activity during its reaction on duplex DNA termini. Addition of high concentrations of nonspecific polymers reverses or prevents this inactivation. In contrast, additions of related materials of lower molecular weight are relatively ineffective in stabilizing the kinase. Such a pattern suggests that the stabilizing effects of polymers on kinase activity are due to macromolecular crowding. An effect of crowding on the known tendency of the kinase to undergo oligomerization reactions is consistent with our observations. This effect of polymers on the kinase can be exploited to greatly increase the amount of reaction obtainable on 5'-hydroxyl groups of duplex DNA substrates located at recessed or blunt ends or at "nicks" which are otherwise difficult to effectively phosphorylate or dephosphorylate. Macromolecular crowding was found to cause no striking change in the processivity of the T4 DNA ligase under several reaction conditions. Interactions with the Patent Office in connection with an application on behalf of the Government have been successfully concluded and we await the issuance of a patent based on our previous studies of polymer-stimulated ligation of DNA.

T4多核苷酸激酶在反应过程中迅速失去活性 双链DNA末端。添加高浓度的非特异性 聚合物可以逆转或防止这种失活。相比之下，添加的 相对分子质量较低的相关材料对 稳定肌动蛋白的作用。这样的模式表明，稳定的 聚合物对激酶活性的影响是由于大分子拥挤造成的。 拥挤对已知的激酶发生趋势的影响 齐聚反应与我们的观察结果一致。这一效果 的聚合物可以被利用来极大地增加 双链DNA底物5‘-羟基上可发生的反应 位于凹入的或钝的末端，或位于“凹口”处，否则 难以有效地进行磷酸化或去磷化。 发现大分子拥挤没有引起显著的变化。 T4DNA连接酶在几种反应条件下的加工性。 与专利局的互动与一项关于 代表政府已圆满结束，我们等待 基于我们之前对聚合物刺激的研究颁发的专利 DNA的连接。