Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function.
Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination.
Ristic
Dejan
D
Department of Cell Biology and Genetics, Erasmus Medical Center PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Modesti
Mauro
M
van der Heijden
Thijn
T
van Noort
John
J
Dekker
Cees
C
Kanaar
Roland
R
Wyman
Claire
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D018625:Microscopy, Atomic Force; D009698:Nucleoproteins / Q000648:ultrastructure; D051135:Rad51 Recombinase; D011995:Recombination, Genetic
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D000255:Adenosine Triphosphate; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase