期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1990
卷号：87
期号：23
页码：9123-9127
DOI：10.1073/pnas.87.23.9123
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Plasmid R6K contains inverted repeats of an approximately 100-base-pair sequence separated by 5.5 kilobases. These long inverted repeats (LIRs) occur within the alpha and beta origins of replication and are essential for origin function. In this study, primer-extension analysis of DNA modified in vivo by dimethyl sulfate or KMnO4 revealed that both alpha and beta LIRs acquire similar structural distortions of the DNA helix in a functional R6K replicon. These distortions were not seen in plasmids containing isolated LIR sequences. In the functional replicon, the dimethyl sulfate and KMnO4 hyperreactive sites appear on complementary strands and are located to one side of an internal palindromic sequence within the LIRs. This asymmetry coincides with the primary direction of DNA replication from alpha and beta origins in vivo. We also observed two intermediate structures when certain R6K cis- or trans-acting elements are missing. Sequences near the alpha origin are required for generation of the dimethyl sulfate hyperreactive sites, whereas sequences near the beta origin are responsible for the appearance of KMnO4 hyperreactive sites. We suggest that these structures represent a hierarchy that leads to a "locked" preinitiation complex, which functions to synchronize and determine the direction of replication from the alpha and beta replication origins in vivo.
