Structural transitions within human Rad51 nucleoprotein filaments.
Rad51 is a core component of the eukaryotic homologous recombination machinery and is responsible for key mechanistic steps during strand invasion. Higher order oligomers of Rad51 display a remarkable degree of structural variation, forming rings, compressed filaments, and elongated filaments. It is unclear whether Rad51 can transition directly between these different oligomeric structures without disassembling first into monomers. We have used single-molecule microscopy to investigate the behavior of human Rad51 assembled on double-stranded DNA. Our results show that human Rad51 can form elongated nucleoprotein filaments on DNA, but ATP hydrolysis causes a decrease in their length without concomitant dissociation of protein. Compressed Rad51 filaments can re-elongate when presented with either ATP or the non-hydrolyzable analog AMP-PNP, and these cycles of elongation and compression are reversible. A Rad51 mutant deficient in ATP hydrolysis is locked into an extended conformation that is incapable of transitioning to a compressed filament. Similarly, wild-type Rad51 bound to DNA in the presence of AMP-PNP was trapped in the elongated state. Proteins incapable of transitioning to the compressed state were also highly resistant to dissociation from the DNA. Taken together, our results indicate that nucleotide hydrolysis by human Rad51 triggers a reversible structural transition leading to filaments with reduced helical pitch.
Robertson
Ragan B
RB
Department of Biological Sciences and Biochemistry, Columbia University, 650 West 168th Street, New York, NY 10032, USA.
Moses
Dana N
DN
Kwon
YoungHo
Y
Chan
Pamela
P
Chi
Peter
P
Klein
Hannah
H
Sung
Patrick
P
Greene
Eric C
EC
D000255:Adenosine Triphosphate / Q000737:chemistry; D002118:Calcium / Q000502:physiology; D004247:DNA / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008856:Microscopy, Fluorescence; D011487:Protein Conformation; D051135:Rad51 Recombinase / Q000737:chemistry*; D012965:Sodium Chloride / Q000494:pharmacology
D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D002118:Calcium