Rad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. cellular metabolism like oxygen radicals, and exogenous sources, including ultraviolet and ionizing radiation (Friedberg et al., 2004). Among different kinds of lesions, DNA double-strand breaks (DSBs) present a special challenge to the cells because both strands of the double helix are affected. If misrepaired, DSBs can cause genome rearrangements such as translocations and deletions that can result in development of cancer (Hoeijmakers, 2001; Bassing and Alt, 2004; Agarwal et al., 2006). Thus, it is paramount that DSBs are repaired precisely and in a timely fashion. Homologous recombination is an error free, high-fidelity pathway that repairs DSBs by using an undamaged homologous DNA molecule, usually the sister chromatid, Trdn as a template to repair the broken molecule (Wyman and Kanaar, 2006). The process is performed by the Rad52 epistasis group proteins, identified by the genetic analyses of ionizing radiationCsensitive mutants (Game and Mortimer, 1974; Symington, Vernakalant Hydrochloride supplier 2002). Several Rad52 group proteins, including Rad51 and Rad54, are conserved in mammals, as is the core mechanism of homologous recombination (Wyman and Kanaar, 2004). The central protein of homologous recombination is Rad51, which mediates the critical step of homologous pairing and DNA strand exchange between the broken DNA molecule and the homologous intact repair template. Once a DSB occurs, it is processed to single-stranded DNA tails with a 3 polarity, onto which Rad51 promoters assemble into a nucleoprotein filament. This nucleoprotein filament is the active molecular entity in recognition of homologous DNA and the subsequent exchange of DNA strands. An extensive number of mediator and/or accessory proteins are implicated in assisting Rad51 at various stages of recombination (Sung et al., 2003), one of which is Rad54. locus. A targeting construct, consisting of the human cDNA exons IVCXVIII fused to a GFP coding sequence or containing a point mutation in the Walker A ATPase domain (Fig. 1 A), was electroporated into ES cells of the genotype allele is inactivated (Tan et al., 1999). Two different mutant constructs were used, one in which the lysine at position 189 was replaced by arginine, which is indicated by K189R and one in which the lysine is replaced by alanine, the K189A mutation. The ATPase activity of the purified Rad54K189R and Rad54K189A proteins was reduced >100-fold in comparison to the wild-type protein (Swagemakers et al., 1998 and unpublished data). Clones carrying a homologously integrated knockin construct were identified by DNA blot analysis. A probe that detects exons VII and VIII was used in combination with genomic DNA digested with StuI, which yielded the expected doublet of bands 6.5 kb for the knockin allele, whereas Vernakalant Hydrochloride supplier a 6.0-kb band was observed that is diagnostic for the knockout allele (Fig. 1 B). Proper expression of the full-length wild-type or mutant Rad54CGFP fusion proteins was confirmed by immunoblot analysis (Fig. 1 C). In the subsequent studies, two independent clones for Vernakalant Hydrochloride supplier and one for were used. As a positive control for all experiments, knockin ES cells were used; these cells express wild-type Rad54 fused to GFP from the endogenous locus. The function of Rad54 is not affected by its fusion to GFP because cells are not DNA damage.