Observations on template switching during DNA replication through long inverted repeats

Abstract

The bacterial Tn5 transposon consists of a pair of long inverted repeats flanking a central region that carries genes for antibiotic resistance. An analysis of DNA replication through Tn5 by two-dimensional gel electrophoresis has revealed two interesting features: (i) a spike representing X-shaped molecules, and (ii) a spot representing a barrier on the arc of Y-shaped replication intermediates. The electrophoretic behavior of various restriction fragments derived from this region indicates that the X molecules contain two linear Tn5 fragments joined together by a cross connection (Holliday junction). However, their formation is recA independent. The junction seems to connect the right inverted repeat of one fragment to the left inverted repeat of the other. The structure of the X molecules suggests that they could be formed by template switching when synthesis of the leading strand enters the right inverted repeat. One possible mechanism is that switching occurs at the center of a transient cruciform structure. A similar switching event, occurring when synthesis of the leading strand enters the left inverted repeat, could give rise to the barrier. These results imply that the inviability of palindromic DNA, which has previously been attributed to the slowing down of replication, may actually be caused by frequent template switching.