Murphy L D, Zimmerman S B
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Biophys Chem. 1995 Dec;57(1):71-92. doi: 10.1016/0301-4622(95)00047-2.
DNA added to concentrated extracts of Escherichia coli undergoes a reversible transition to a readily-sedimentable ('condensed') form. The transition occurs over a relatively small increment in extract concentration. The extract appears to play two roles in this transition, supplying both DNA-binding protein(s) and a crowded environment that increases protein binding and favors compact DNA conformations. The two roles of the extract are suggested by properties of fractions prepared by absorption of extracts with DNA-cellulose. The DNA-binding fraction and the DNA-nonbinding fractions from these columns are separately poorer condensing agents than the original extract, but when rejoined are similar to the original extract in the amount required for condensation. The dual role for the extract is supported by model studies of condensation with combinations of purified DNA-binding materials (protein HU or spermidine) and concentrated solutions of crowding agents (albumin or polyethylene glycol 8000); in each case, crowding agents and DNA-binding materials jointly reduce the amounts of each other required for condensation. The condensation reaction as studied in extracts or in the purified systems may be a useful approach to the forces which stabilize the compact form of DNA within the bacterial nucleoid. The effect of condensation on the reactivity of the DNA was measured by changes in the rate of cohesion between duplex DNA molecules bearing the complementary single-strand termini of lambda DNA. Condensation caused large increases in the rates of cohesion of both lambda DNA and of restriction fragments of lambda DNA bearing the cohesive termini. Cohesion products of lambda DNA made in vitro are a mixture of linear and circular aggregates, whereas those made in vivo are cyclic monomers. We suggest a simple mechanism based upon condensation at the site of viral injection which may explain this discrepancy.
添加到大肠杆菌浓缩提取物中的DNA会发生可逆转变，形成易于沉降的（“浓缩”）形式。这种转变在提取物浓度相对较小的增量范围内发生。提取物在这种转变中似乎起到了两个作用，既提供了DNA结合蛋白，又提供了一个拥挤的环境，这种环境增加了蛋白结合并有利于紧密的DNA构象。提取物的这两个作用是由用DNA-纤维素吸附提取物制备的各组分的性质所表明的。来自这些柱的DNA结合组分和DNA非结合组分作为凝聚剂分别比原始提取物差，但重新组合后在凝聚所需的量方面与原始提取物相似。提取物的双重作用得到了用纯化的DNA结合材料（蛋白HU或亚精胺）和拥挤剂（白蛋白或聚乙二醇8000）的组合进行凝聚模型研究的支持；在每种情况下，拥挤剂和DNA结合材料共同减少了彼此凝聚所需的量。在提取物或纯化系统中研究的凝聚反应可能是了解稳定细菌类核内DNA紧密形式的力的一种有用方法。通过带有λDNA互补单链末端的双链DNA分子之间的凝聚速率变化来测量凝聚对DNA反应性的影响。凝聚导致λDNA以及带有粘性末端的λDNA限制性片段的凝聚速率大幅增加。体外制备的λDNA凝聚产物是线性和环状聚集体的混合物，而体内制备的则是环状单体。我们提出了一种基于病毒注射部位凝聚的简单机制，这可能解释了这种差异。
