Sun M, Son M, Serwer P
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78284-7760, USA.
Biochemistry. 1997 Oct 21;36(42):13018-26. doi: 10.1021/bi971410b.
To understand in vivo DNA metabolism, in vitro systems are developed that perform DNA metabolism, while maintaining in vivo (physiological) character. To determine the state of DNA during in vitro physiological metabolism, the present study develops procedures of fluorescence light microscopy for observation of stained DNA molecules during in vitro physiological metabolism in a crude extract of bacteriophage T7-infected cells. The extract inhibits illumination-induced breakage of DNA. The following DNA metabolism remains active for 2-3 min during microscopy: exonuclease-dependent end-to-end joining (concatemerization) of T7 DNA and subsequent cleavage of concatemers. When the T7 gene 3-encoded DNA debranching endonuclease is absent during in vitro T7 DNA concatemerization, DNA progressively partitions to form a continuous, mostly immobile (i.e., no detected Brownian motion) fibrous network that encloses the DNA-depleted solution; presumably because of reduced branching, a less extensive network forms when the gene 3-encoded debranching endonuclease is present. Most strands of the network consist of multiple DNA segments. After a time interval of 5-10 min, the DNA network undergoes cleavage that depends on the presence of both ATP, capsids, and the DNA packaging accessory proteins encoded by genes 18 and 19; multiple cleavages eventually disrupt the continuity of the DNA network. The dependence of the observed cleavage on these factors is explained by the hypothesis that this cleavage is the first of two cleavages known to occur during the packaging of T7 DNA concatemers both in vivo and in vitro. The first cleavage is also known to initiate entry of DNA into a T7 capsid. The cleavage observed here is usually preceded by an approximately 10 s burst of oscillatory motion of the DNA network near the point of eventual cleavage. If the in vivo presence of a similar concatemer-containing DNA network is assumed, requirement for DNA packaging-associated release of DNA from this network is a possible explanation for the evolution of a T7 DNA packaging pathway that is initiated by cleavage of a concatemer.
为了理解体内DNA代谢，人们开发了体外系统来进行DNA代谢，同时保持体内（生理）特性。为了确定体外生理代谢过程中DNA的状态，本研究开发了荧光显微镜观察程序，用于观察噬菌体T7感染细胞粗提物中体外生理代谢过程中染色的DNA分子。该提取物可抑制光照诱导的DNA断裂。在显微镜观察期间，以下DNA代谢在2 - 3分钟内保持活跃：T7 DNA的核酸外切酶依赖性端对端连接（多联体形成）以及随后多联体的切割。当在体外T7 DNA多联体形成过程中不存在T7基因3编码的DNA去分支内切酶时，DNA逐渐分区形成一个连续的、大多固定不动（即未检测到布朗运动）的纤维网络，该网络包围着DNA耗尽的溶液；据推测，由于分支减少，当存在基因3编码的去分支内切酶时会形成不太广泛的网络。该网络的大多数链由多个DNA片段组成。在5 - 10分钟的时间间隔后，DNA网络会发生切割，这取决于ATP、衣壳以及基因18和19编码的DNA包装辅助蛋白的存在；多次切割最终会破坏DNA网络的连续性。观察到的切割对这些因素的依赖性可以通过以下假设来解释，即这种切割是已知在体内和体外T7 DNA多联体包装过程中发生的两次切割中的第一次。第一次切割也已知会启动DNA进入T7衣壳。这里观察到的切割通常之前会有大约10秒的DNA网络在最终切割点附近的振荡运动爆发。如果假设体内存在类似的含多联体DNA网络，那么对DNA包装相关的从该网络释放DNA的需求可能是T7 DNA包装途径由多联体切割启动这一进化过程的一种解释。
