Metnase, also known as SETMAR, is a SET and transposase fusion protein with an undefined role in mammalian DNA repair. The SET domain is responsible for histone lysine methyltransferase activity at histone 3 K4 and K36, whereas the transposase domain possesses 5-terminal inverted repeat (TIR)-specific DNA binding, DNA looping, and DNA cleavage activities. Although the transposase domain is essential for Metnase function in DNA repair, it is not clear how a protein with sequencespecific DNA binding activity plays a role in DNA repair. Here, we show that human homolog of the ScPSO4/PRP19 (hPso4) forms a stable complex with Metnase on both TIR and non-TIR DNA. The transposase domain essential for Metnase-TIR interaction is not sufficient for its interaction with non-TIR DNA in the presence of hPso4. In vivo, hPso4 is induced and co-localized with Metnase following ionizing radiation treatment. Cells treated with hPso4-siRNA failed to show Metnase localization at DSB sites and Metnase-mediated stimulation of DNA end joining coupled to genomic integration, suggesting that hPso4 is necessary to bring Metnase to the DSB sites for its function(s) in DNA repair.Metnase (SETMAR) is a double strand break (DSB) 2 repair factor that contains two functional domains: a SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain of histone lysine methyltransferase activity at histone 3, lysine 4, and lysine 36 (1) associated with chromatin opening (2-5) and a transposase domain containing the DDE motif (1, 6 -8), a conserved acidic motif essential for strand transfer and end joining activities among transposase and retroviral integrase families (9 -13). Deletion of either SET or transposase domain abolished Metnase function in vivo (1), suggesting that both domains are likely required for its role in DSB repair and genomic integration. Although Metnase is not an active transposase, it possesses most transposase functions such as sequence-specific DNA binding (6,11,14), assembly of paired end complexes (14), and DNA cleavage activity (11,14). Unlike other transposases, however, Metnase-mediated DNA cleavage was nonprocessive and occurred in the absence of the TIR sequence (11).Human Pso4 (hPso4) is a human homolog of the protein encoded by the PS04/PRP19 gene in Saccharomyces cerevisiae (15,16). PSO4 gene is essential for cell survival in yeast (15), and cells harboring a mutant Pso4 showed sensitivity to DNA crosslinking agents, suggesting that PSO4 is an essential DNA repair gene in S. cerevisiae (15). Pso4 is a part of the pre-mRNA splicing complex consisting of Pso4, Cdc5L, Plrg1, and Spf27 (17) and has been previously linked to DNA repair through a direct physical interaction between Cdc5L and WRN, the protein deficient in Werner syndrome (18). Human Pso4 contains six successive WD-40 motifs at the C terminus that is known to form a structural interface for the assembly of multiprotein complexes (19) and has been identified as a component of the nuclear matrix (20). Pso4 is also a U-box protein with associated E3 ubiquitin ligase...