Specific fragmentation of T7 phage DNA at low-melting sites.

A method has been developed for selective fragmentation of T7 DNA at AT-rich regions. The molecules have been subjected to complete digestion with single-strand-specific SI endonuclease after fixation of DNA AT-rich regions in the denatured state by glyoxal. The treatment resulted in three fragments having molecular weights of 13.6 +/- 0.4, 8.2 +/- 0.4 and 3.5 +/- 0.16 megadaltons as determined by electron microscopy. The position of these fragments along the T7 DNA molecule has been determined by means of analysis of the intermediates during SI-cleavage

Mechanisms of triplex-caused polymerization arrest.

Pyrimidine/purine/purine triplexes are known to inhibit DNA polymerization. Here we have studied the mechanisms of this inhibition by comparing the efficiency of Vent DNA polymerase on triplex- and duplex-containing templates at different temperatures, Mg2+concentrations and time intervals with the thermal stability of the corresponding structures. Our results show that triplexes can only be by-passed at temperatures where thermal denaturation initiates, while duplexes, in contrast, are overcome at temperatures where they are quite stable. These results show that DNA polymerase cannot untangle triplex regions within DNA templates and seems to entirely depend on their thermal fluctuations. The high stability of triplexes at physiological temperatures and ambient conditions make them a barrier to polymerization