Base-pair substitutions in avian sarcoma virus U5 and U3 long terminal repeat sequences alter the process of DNA integration in vitro.
Base-pair substitutions in avian sarcoma virus U5 and U3 long terminal repeat sequences alter the process of DNA integration in vitro.
复制标题
禽肉瘤病毒 U5 和 U3 长末端重复序列中的碱基对取代改变了体外 DNA 整合的过程。
DOI：
10.1128/jvi.75.3.1132-1141.2001
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发表时间：
2001
影响因子：
5.4
通讯作者：
Leis,J
中科院分区：
文献类型：
--
作者：
Hindmarsh,P;Johnson,M;Reeves,R;Leis,J
我们描述了一个重组的禽肉瘤病毒（ASV）协同DNA整合系统，该系统包括一个特殊设计的含有upF转录单位的微型供体DNA、一个超螺旋质粒受体、纯化的细菌表达的ASV整合酶（IN）和人高迁移率族蛋白I（Y）。在该系统中的整合取决于在两端具有IN识别序列的微型供体DNA和相同供体的两端整合到受体DNA中。整合的DNA产物表现出与病毒DNA体内整合相关的所有特征（P. Hindmarsh等，J. Virol.，73：2994-3003，1999）。从含有具有琥珀突变的耐药标志物的细菌中分离单个整合体。该系统用于评估在邻近保守CA二核苷酸的位置5和/或6处的末端U 5和U3长末端重复序列中的序列的重要性。在U 5中的这些位置处引入的碱基对取代导致从细菌回收的整合体的显著减少，这是由于单端插入事件的增加。在从与突变的U 5而不是U3 IN识别序列的反应中回收的整合体中，是在受体DNA中含有大缺失的产物。U3中位置5和6处的碱基对取代主要降低经修饰的供体的整合效率。总之，这些结果表明，直接5′端保守的CA二核苷酸序列是非常重要的协同DNA整合的过程。此外，IN与U3和U 5末端的相互作用不同，并且导致非协调DNA整合的异常末端加工事件在U 5中比在U3中更常见。
We have described a reconstituted avian sarcoma virus (ASV) concerted DNA integration system with specially designed mini-donor DNA containing asupFtranscription unit, a supercoiled plasmid acceptor, purified bacterially expressed ASV integrase (IN), and human high-mobility-group protein I(Y). Integration in this system is dependent upon the mini-donor DNA having IN recognition sequences at both ends and upon both ends of the same donor integrating into the acceptor DNA. The integrated DNA product exhibits all of the features associated with integration of viral DNA in vivo (P. Hindmarsh et al., J. Virol., 73:2994–3003, 1999). Individual integrants are isolated from bacteria containing drug-resistant markers with amber mutations. This system was used to evaluate the importance of sequences in the terminal U5 and U3 long terminal repeats at positions 5 and/or 6, adjacent to the conserved CA dinucleotide. Base-pair substitutions introduced at these positions in U5 result in significant reductions in recovered integrants from bacteria, due to increases in one-ended insertion events. Among the recovered integrants from reactions with mutated U5 but not U3 IN recognition sequences were products that contain large deletions in the acceptor DNA. Base-pair substitutions at positions 5 and 6 in U3 mostly reduce the efficiency of integration of the modified donor. Together, these results indicate that sequences directly 5′ to the conserved CA dinucleotide are very important for the process of concerted DNA integration. Furthermore, IN interacts with U3 and U5 termini differently, and aberrant end-processing events leading to nonconcerted DNA integration are more common in U5 than in U3.