Direct observation of DNA rotation during branch migration of Holliday junction DNA by Escherichia coli RuvA-RuvB protein complex.
The Escherichia coli RuvA-RuvB complex promotes branch migration of Holliday junction DNA, which is the central intermediate of homologous recombination. Like many DNA motor proteins, it is suggested that RuvA-RuvB promotes branch migration by driving helical rotation of the DNA. To clarify the RuvA-RuvB-mediated branch migration mechanism in more detail, we observed DNA rotation during Holliday junction branch migration by attaching a bead to one end of cruciform DNA that was fixed to a glass surface at the opposite end. Bead rotation was observed when RuvA, RuvB, and ATP were added to the solution. We measured the rotational rates of the beads caused by RuvA-RuvB-mediated branch migration at various ATP concentrations. The data provided a K(m) value of 65 microM and a V(max) value of 1.6 revolutions per second, which corresponds to 8.3 bp per second. This real-time observation of the DNA rotation not only allows us to measure the kinetics of the RuvA-RuvB-mediated branch migration, but also opens the possibility of elucidating the branch migration mechanism in detail.
Han
Yong-Woon
YW
Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan.
Tani
Tomomi
T
Hayashi
Masahito
M
Hishida
Takashi
T
Iwasaki
Hiroshi
H
Shinagawa
Hideo
H
Harada
Yoshie
Y
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D045566:DNA, Cruciform / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes; D009690:Nucleic Acid Conformation
D001426:Bacterial Proteins; D045566:DNA, Cruciform; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; C061260:RuvB protein, Bacteria; D000255:Adenosine Triphosphate; D004247:DNA; C071059:Holliday junction DNA helicase, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases