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 Å per base pair) and untwisted by 18.2°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°؎ 5°. 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.homologous recombination ͉ magnetic tweezers ͉ nucleation and growth ͉ single molecule ͉ mechanoenzyme H omologous recombination (HR) is the main pathway for accurate repair of DNA double-strand breaks and maintenance of genome integrity. It is also essential for overcoming stalled replication forks. Rad51 is the key protein of HR and the recombinational DNA repair process in eukaryotes (1-3). First, Rad51 polymerizes at or near one end of a DNA break, forming a right-handed helical nucleoprotein filament. This nucleoprotein filament then searches the genome for a homologous DNA sequence. Once the nucleoprotein filament and homologous sequence are synapsed, new DNA synthesis can proceed using the homologous sequence as template. Finally, this structure is resolved to repair the DNA break. Despite intense efforts, some divergences remain about the formation of the nucleoprotein filament. Nucleoprotein filaments assembled onto ssDNA or dsDNA in the presence of ATP as a cofactor display very similar helical structures on electron microscopy (EM), with a rise per base pair of 4.7 Å (4). This structure also looks much like that achieved with RecA, the bacterial homologue of Rad51 (5). The stoichiometry of both proteins has been shown to depend on experimental conditions (6). Reported values range from 3 to 7 bp per protein for RecA (5, 7-9) and 2 to 3 and 4 to 5 bp per protein for Rad51 (4, 10-12). The kinetics of RecA and Rad51 polymerization were recently the subject of several singlemolecule studies (13-21). There is consensus about a nucleation and growth mechanism for both RecA (14-16, 22) and Rad51 (19-21), and abo...