Analysis of mouse Rad54 expression and its implications for homologous recombination.
Homologous recombination is one of the major pathways for repair of DNA double-strand breaks (DSBs). Important proteins in this pathway are Rad51 and Rad54. Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) that mediates pairing with and strand invasion of homologous duplex DNA with the assist of Rad54. We estimated that the nucleus of a mouse embryonic stem (ES) cells contains on average 4.7x10(5) Rad51 and 2.4x10(5) Rad54 molecules. Furthermore, we showed that the amount of Rad54 was subject to cell cycle regulation. We discuss our results with respect to two models that describe how Rad54 stimulates Rad51-mediated DNA strand invasion. The models differ in whether Rad54 functions locally or globally. In the first model, Rad54 acts in cis relative to the site of strand invasion. Rad54 coats the Rad51 nucleoprotein filament in stoichiometric amounts and binds to the target duplex DNA at the site that is homologous to the ssDNA in the Rad51 nucleoprotein filament. Subsequently, it promotes duplex DNA unwinding. In the second model, Rad54 acts in trans relative to the site of strand invasion. Rad54 binds duplex DNA distant from the site that will be unwound. Translocation of Rad54 along the duplex DNA increases superhelical stress thereby promoting duplex DNA unwinding.
Essers
Jeroen
J
Department of Cell Biology and Genetics, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
Hendriks
Rudi W
RW
Wesoly
Joanna
J
Beerens
Cecile E M T
CE
Smit
Bep
B
Hoeijmakers
Jan H J
JH
Wyman
Claire
C
Dronkert
Mies L G
ML
Kanaar
Roland
R
D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases; D004260:DNA Repair / Q000502:physiology*; D045643:DNA Repair Enzymes; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli; D005434:Flow Cytometry; D015151:Immunoblotting; D017403:In Situ Hybridization; D051379:Mice; D039904:Pluripotent Stem Cells / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*
D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes