环境对大肠杆菌体内分子内三链DNA水平的影响。

Environmental influences on the in vivo level of intramolecular triplex DNA in Escherichia coli.

作者信息

Ussery D W, Sinden R R

机构信息

Institute of Biosciences and Technology, Texas A&M University, Houston 77030-3303.

出版信息

Biochemistry. 1993 Jun 22;32(24):6206-13. doi: 10.1021/bi00075a013.

DOI:10.1021/bi00075a013

PMID:8512930

Abstract

We describe an assay for detecting intramolecular triple-stranded DNA in living cells. The assay involves quantitative analysis of the differential photobinding of 4,5',8-trimethylpsoralen to 5'TA and 5'AT dinucleotides in a region of plasmid DNA containing the triplex-forming sequence (GA)7TA(GA)7. Psoralen photobinds to the central 5'TA within the (GA)7TA(GA)7 sequence in duplex DNA but not when the sequence exists as an intramolecular triplex structure. The reactivity of photobinding sites in regions flanking the intramolecular triplex-forming sequence, those that comprise the duplex-triplex junctions, either increase or decrease upon formation of the intramolecular triplex structure from duplex DNA. The pattern of trimethylpsoralen reactivity provides an indication of the conformation of the (GA)7TA(GA)7 sequence in plasmid DNA. The formation of the intramolecular triplex structure is dependent on both pH and negative superhelical density in vitro. The fraction of the (GA)7TA(GA)7 sequence that existed as an intramolecular triplex structure in vivo was dependent on the level of DNA supercoiling in vivo and changes in growth conditions which influence the intracellular pH. The Hy3 isomer was detected in living Escherichia coli cells.

摘要

我们描述了一种用于检测活细胞内分子内三链DNA的分析方法。该分析方法涉及对4,5',8-三甲基补骨脂素与质粒DNA中包含三链形成序列（GA）7TA（GA）7区域内的5'TA和5'AT二核苷酸的差异光结合进行定量分析。补骨脂素在双链DNA中的（GA）7TA（GA）7序列内与中央5'TA发生光结合，但当该序列以分子内三链结构存在时则不会发生光结合。分子内三链形成序列侧翼区域（即构成双链-三链连接的区域）中光结合位点的反应性，在双链DNA形成分子内三链结构时会增加或降低。三甲基补骨脂素的反应模式表明了质粒DNA中（GA）7TA（GA）7序列的构象。分子内三链结构的形成在体外取决于pH值和负超螺旋密度。在体内，（GA）7TA（GA）7序列以分子内三链结构存在的比例取决于体内DNA超螺旋水平以及影响细胞内pH值的生长条件变化。在活的大肠杆菌细胞中检测到了Hy3异构体。