KILOBASE-RANGE COMMUNICATION BETWEEN POLYPURINE-CENTER-DOT-POLYPYRIMIDINE TRACTS IN LINEAR PLASMIDS MEDIATED BY TRIPLEX FORMATION - A BRAIDED KNOT BETWEEN 2 LINEAR DUPLEXES

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 t ethered loop could also be formed in a linear plasmid containing two i dentical tracts by adding an homologous single-stranded oligopyrimidin e, but not an oligopurine. In plasmids containing different tracts, th e formation of both dimers and loops could be blocked by adding either homologous single-stranded oligopyrimidine but not an oligopurine. To gether with the requirement of low pH, these results demonstrate that tripler formation is of the pyr.pur.pyr type. The extent of dimer and loop formation was dependent on the length of the pyrimidine tract; di mers could be detected in plasmids containing the 72 base pair (bp) in serts after incubation at pH 6, but in plasmids containing the 36 bp i nserts, a pH of 5 was required. Hysteresis was also evident to a remar kable extent. Once formed at pH 4, loops and dimers remained stable in definitely at pH 8, suggesting that the structures become topologicall y trapped. However, the structures were resolved into the component li near plasmids by incubation with nuclease P1. This is the first demons tration of a braided or hydrogen-bonded knot between two linear duplex es and may have implications for chromosomal loop formation.