Counting RAD51 proteins disassembling from nucleoprotein filaments under tension.
The central catalyst in eukaryotic ATP-dependent homologous recombination consists of RAD51 proteins, polymerized around single-stranded DNA. This nucleoprotein filament recognizes and invades a homologous duplex DNA segment. After strand exchange, the nucleoprotein filament should disassemble so that the recombination process can be completed. The molecular mechanism of RAD51 filament disassembly is poorly understood. Here we show, by combining optical tweezers with single-molecule fluorescence microscopy and microfluidics, that disassembly of human RAD51 nucleoprotein filaments results from the interplay between ATP hydrolysis and the release of the tension stored in the filament. By applying external tension to the DNA, we found that disassembly slows down and can even be stalled. We quantified the fluorescence of RAD51 patches and found that disassembly occurs in bursts interspersed by long pauses. After relaxation of a stalled complex, pauses were suppressed resulting in a large burst. These results indicate that tension-dependent disassembly takes place only from filament ends, after tension-independent ATP hydrolysis. This integrative single-molecule approach allowed us to dissect the mechanism of this principal homologous recombination reaction step, which in turn clarifies how disassembly can be influenced by accessory proteins.
van Mameren
Joost
J
Laser Centre and Department of Physics and Astronomy, VU University, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.
Modesti
Mauro
M
Kanaar
Roland
R
Wyman
Claire
C
Peterman
Erwin J G
EJ
Wuite
Gijs J L
GJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D006868:Hydrolysis; D044085:Microfluidics; D008856:Microscopy, Fluorescence; D052898:Optical Tweezers; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000378:metabolism*; D011995:Recombination, Genetic; D013997:Time Factors
D004279:DNA, Viral; D000255:Adenosine Triphosphate; C495153:RAD51 protein, human; D051135:Rad51 Recombinase