Kilobase-range communication between polypurine.cntdot.polypyrimidine tracts in linear plasmids mediated by triplex formation: a braided knot between two linear duplexes

Abstract

Linear plasmids were constructed containing two pyrimidine tracts that were 0.34 and 0.94 kilobases (kb) from either end and were separated by 2.8 kb. The tracts [d(TCCTTC)n and d(CTTCCT)n where n = 6 or 12] were designed so as to be able to form triplexes with each other but not with themselves. Upon lowering of the pH to 4 in the presence of spermine, these plasmids form intermolecular dimers and intramolecular loops of 2.8 kb, as judged from mobility changes on agarose gels. A tethered loop could also be formed in a linear plasmid containing two identical tracts by adding an homologous single-stranded oligopyrimidine, but not an oligopurine. In plasmids containing different tracts, the formation of both dimers and loops could be blocked by adding either homologous single-stranded oligopyrimidine but not an oligopurine. Together with the requirement of low pH, these results demonstrate that triplex formation is of the pyr.pur.pyr type. The extent of dimer and loop formation was dependent on the length of the pyrimidine tract: dimers could be detected in plasmids containing the 72 base pair (bp) inserts after incubation at pH 6, but in plasmids containing the 36 bp inserts, a pH of 5 was required. Hysteresis was also evident to a remarkable extent. Once formed at pH 4, loops and dimers remained stable indefinitely at pH 8, suggesting that the structures become topologically trapped. However, the structures were resolved into the component linear plasmids by incubation with nuclease P1. This is the first demonstration of a braided or hydrogen-bonded knot between two linear duplexes and may have implications for chromosomal loop formation.