Direct observation of twisting steps during Rad51 polymerization on DNA

作者：Arata Hideyuki; Dupont Aurelie; Mine Hattab Judith; Disseau Ludovic; Renodon Corniere Axelle; Takahashi Masayuki; Viovy Jean Louis^*; Cappello Giovanni

来源：Proceedings of the National Academy of Sciences, 2009, 106(46): 19239-19244.

DOI：10.1073/pnas.0902234106

• homologous recombination
• magnetic tweezers
• nucleation and growth
• single molecule
• mechanoenzyme

The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5. Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 angstrom per base pair) and untwisted by 18.2 degrees per base pair. They also confirm a stoichiometry of 3-4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65 degrees +/- 5 degrees. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.

• 出版日期2009-11-17