Triple-helical DNA as a reversible block of the branch point in a partially symmetrical DNA four-arm junction.
DNA branch migration is a fundamental process in genetic recombination. A new model system has been developed for studying branch migration in a small synthetic four-arm junction. A mathematical method for describing branch-point movement by discrete steps in such junctions is also presented. The key to our experimental system is the ability to fix the location of the branch point during the assembly of the junction with a reversible block. The block is provided by a short oligonucleotide that forms triplex DNA adjacent to the initial location branch point at low pH. Raising the pH causes the triplex strand to dissociate, making the branch point free to migrate. Once mobile, the branch point can run off the end of the junction. The time-course for this runoff is consistent with a random walk of the branch point. If it is assumed that one migration step moves the branch point one base-pair, the time-course gives a rate constant for one step of 1.4 second-1 at 37 degrees C in 10 mM MgCl2, 50 mM NaCl. These values are consistent with other measurements of non-enzymatic branch migration. We have also monitored the spread of the branch points directly with T4 endonuclease VII. Using EcoRI restriction endonuclease, we have shown that the binding of this protein to the arms of the junction essentially blocks branch migration through the binding site. In these experiments Ca2+ replaces Mg2+, and the enzyme does not cleave the DNA. In vivo there must be a special process to get branch points to migrate past bound proteins.
Kirby
A W
AW
Department of Chemistry, Vassar College, Poughkeepsie, NY 12601, USA.
Gaskin
M N
MN
Antezana
M A
MA
Goodman
S J
SJ
Myers
E
E
Bruist
M F
MF
D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases / Q000378:metabolism; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D013997:Time Factors
D004268:DNA-Binding Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII