Unrepaired double-strand breaks (DSBs) disrupt the integrity of DNA and may lead to genome instability, which has been linked to aging and many human diseases, including cancer. In eukaryotic cells, DSBs are repaired primarily by non-homologous end-joining or homologous recombination (HR). The central protein of HR is Rad51 which, in conjunction with multiple auxiliary proteins, facilitates the initial steps of HR which include the homology searching and strand invasion steps that prime for repair synthesis (3’ polymerization). In this study, the early steps of HR were investigated in mouse hybridoma cells by monitoring kinetics of 3’ polymerization, using a PCR-based assay that detects the repair of a plasmid-borne double-strand gap using a cognate chromosomal gene. Overexpression of wild-type (wt) Rad51 stimulates 3’ polymerization frequency but not the tract length, and notably, increased wtRad51 concentration and homology-length interact synergistically to promote 3’ polymerization. In contrast, a significant DNA repair synthesis defect is observed in cell lines stably expressing Rad51 catalytic variants that are deficient in ATP binding and hydrolysis. Expression of ATP binding-deficient Rad51 is more toxic and increases sensitivity to sequence heterology during 3’ polymerization. Further, expression of the Rad51 catalytic variants accelerates proteasome- mediated depletion of endogenous mouse Rad51 and BRCA2 (Breast Cancer susceptibility 2), and the ensuing deficit in HR selects for low levels of p53 that promote cell survival. The 3’ polymerization process is stimulated by moderate overexpression of human BRCA2 (hBRCA2) but significantly reduced by shRNA-mediated depletion of mouse BRCA2. Expression of wt-hBRCA2, but not a hBRCA2 variant devoid of BRC repeats 1-8, rescues the 3’ polymerization defect in the BRCA2-depleted cells which highlights the role of the BRC repeats in promoting HR. 3’ polymerization is also stimulated by excess Rad54 but inhibited by shRNA-mediated depletion of Rad54 or the expression of Rad54 catalytic variants that are defective in ATP binding or hydrolysis. Notably, wtRad54 overexpression does not rescue the 3’ polymerization defect in Rad51 shRNA-depleted cells or in cells expressing Rad51 catalytic variants, suggesting that Rad54 promotes early events of HR by interacting with recombinationally active Rad51 nucleoprotein filaments.