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.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair Enzymes/metabolism , DNA Repair/physiology , DNA-Binding Proteins/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Nuclear Proteins/metabolism , Terminal Repeat Sequences/physiology , Cell Line , DNA Repair Enzymes/antagonists & inhibitors , DNA Repair Enzymes/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Histone-Lysine N-Methyltransferase/genetics , Histones/metabolism , Humans , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Protein Structure, Tertiary/physiology , RNA Splicing Factors , RNA, Small Interfering/geneticsABSTRACT
Metnase (SETMAR) is a SET and transposase fusion protein that promotes in vivo end joining activity and mediates genomic integration of foreign DNA. Recent studies showed that Metnase retained most of the transposase activities, including 5'-terminal inverted repeat (TIR)-specific binding and assembly of a paired end complex, and cleavage of the 5'-end of the TIR element. Here we show that R432 within the helix-turn-helix motif is critical for sequence-specific recognition, as the R432A mutation abolishes its TIR-specific DNA binding activity. Metnase possesses a unique DNA nicking and/or endonuclease activity that mediates cleavage of duplex DNA in the absence of the TIR sequence. While the HTH motif is essential for the Metnase-TIR interaction, it is not required for its DNA cleavage activity. The DDE-like motif is crucial for its DNA cleavage action as a point mutation at this motif (D483A) abolished its DNA cleavage activity. Together, our results suggest that Metnase's DNA cleavage activity, unlike those of other eukaryotic transposases, is not coupled to its sequence-specific DNA binding.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , Recombinant Fusion Proteins/metabolism , Transcription Factors/metabolism , Transposases/metabolism , Amino Acid Sequence , Base Sequence , Blotting, Western , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , DNA Cleavage , DNA-Binding Proteins , Electrophoresis, Polyacrylamide Gel , Electrophoretic Mobility Shift Assay , Histone Chaperones , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Molecular Sequence Data , Protein Binding , Recombinant Fusion Proteins/chemistry , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/genetics , Transposases/chemistry , Transposases/geneticsABSTRACT
Parthenolide, a sesquiterpene lactone, shows antitumor activity in vitro, which correlates with its ability to inhibit the DNA binding of the antiapoptotic transcription factor nuclear factor kappaB (NF-kappaB) and activation of the c-Jun NH(2)-terminal kinase. In this study, we investigated the chemosensitizing activity of parthenolide in vitro as well as in MDA-MB-231 cell-derived xenograft metastasis model of breast cancer. HBL-100 and MDA-MB-231 cells were used to measure the antitumor and chemosensitizing activity of parthenolide in vitro. Parthenolide was effective either alone or in combination with docetaxel in reducing colony formation, inducing apoptosis and reducing the expression of prometastatic genes IL-8 and the antiapoptotic gene GADD45beta1 in vitro. In an adjuvant setting, animals treated with parthenolide and docetaxel combination showed significantly enhanced survival compared with untreated animals or animals treated with either drug. The enhanced survival in the combination arm was associated with reduced lung metastases. In addition, nuclear NF-kappaB levels were lower in residual tumors and lung metastasis of animals treated with parthenolide, docetaxel, or both. In the established orthotopic model, there was a trend toward slower growth in the parthenolide-treated animals but no statistically significant findings were seen. These results for the first time reveal the significant in vivo chemosensitizing properties of parthenolide in the metastatic breast cancer setting and support the contention that metastases are very reliant on activation of NF-kappaB.
