We are characterizing a key step in genetic recombination, DNA branch migration. Pairing of two homologous DNA duplexes and strand exchange carried out by recombination proteins like E. coli RecA led to the formation of a central intermediate in recombination, the Holliday junction. The Holliday junction, corresponding to the exchange point between two homologous DNAs, can migrate by the step-wise exchange of hydrogen bonds between identical strands of the two DNA duplexes. This process is known as branch migration, and it dictates the extent of transfer of genetic information between two homologous chromosomes. Understanding the mechanics of branch migration is critical to understanding the mechanism of homologous recombination and the role that certain proteins play in promoting branch migration during recombination. We have recently measured the rate of spontaneous or uncatalysed DNA branch migration. In the presence of physiological concentrations of magnesium, branch migration is quite slow with a step time of about 300 msec. However, n the absence of magnesium, branch migration is significantly faster and, depending on the ionic strength, can proceed 1000 to 10,000 times faster than in magnesium. We have initiated studies to examine the effect of subtle changes in the conformation of the Holliday junction on the rate of branch migration. Diethylpyrocarbonate and osmium tetroxide have been used to chemically probe the extent of base stacking at the crossover point in synthetic four-way DNA junctions that mimic the Holliday junction. Our result demonstrate that a small drop in the concentration of magnesium ions, from 300 to 100 uM, results in an abrupt loss of base stacking in the four-way junction; this conformational change induced by lowering the magnesium concentration is parallelled by a large increase (about 30- fold) in the rate of spontaneous branch migration. We conclude that some property of the magnesium-induced, stacked Holliday structure presents a kinetic barrier to branch migration; disruption of base stacking at the crossover point circumvents this slow step. Currently, we are employing both enzymatic and chemical modification techniques to assess the conformational changes in the Holliday junction that occur during branch migration.
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