Abstract
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 sequence-specific 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.
Highlights
Posase, 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]
We show that a damage-induced DNA repair factor, Human Pso4 (hPso4) is the Metnase-binding partner that mediates Metnase interaction with non-terminal inverted repeat (TIR) DNA such as DNA damage sites necessary for the function of Metnase in double strand break (DSB) repair
Specific role(s) for Pso4 in ated Stimulation of DNA End Joining Coupled to Genomic DSB repair is not known; it has been identified as a Integration—Pso4 is a dsDNA-binding protein that plays a component of the nuclear matrix [20]
Summary
Enzymes, and Chemicals—Human embryonic kidney (HEK 293) cells were grown in Dulbecco’s modified Eagle’s. Purification of FLAG-Metnase and FLAG-hPso4—Metnase (or hPso4)-expressing cells (1.6 ϫ 108) were suspended in 20 ml of extraction buffer (TEGDN; 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 5 mM dithiothreitol, 1.0% Nonidet-P40, and mammalian protease inhibitor cocktails containing 0.2 M NaCl) and centrifuged (100,000 ϫ g) for 30 min. The S100 fraction was incubated at 4 °C for 60 min with anti-FLAG M2 affinity gel (Sigma) that had been pre-equilibrated with TEGDN buffer containing 0.2 M NaCl. The beads were washed three times with TEGDN, 2.0 M NaCl buffer prior to elution of the protein with TEGDN, 0.2 M NaCl containing FLAG peptide (500 g/ml).
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