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...

Metnase (SETMAR) is a SET-transposase fusion protein that promotes non-homologous end joining (NHEJ) repair in humans. Although both SET and the transposase domains were necessary for its function in DSB repair, it is not clear what specific role Metnase plays in the NHEJ. In this study, we show that Metnase possesses a unique endonuclease activity that preferentially acts on ssDNA and ssDNA-overhang of a partial duplex DNA. Cell extracts lacking Metnase poorly supported DNA end joining, and addition of wt-Metnase to cell extracts lacking Metnase markedly stimulated DNA end joining, while a mutant (D483A) lacking endonuclease activity did not. Given that Metnase overexpression enhanced DNA end processing in vitro, our finding suggests a role for Metnase's endonuclease activity in promoting the joining of non-compatible ends.

Metnase is a human SET and transposase domain protein that methylates histone H3 and promotes DNA double-strand break repair. We now show that Metnase physically interacts and co-localizes with Topoisomerase IIα (Topo IIα), the key chromosome decatenating enzyme. Metnase promotes progression through decatenation and increases resistance to the Topo IIα inhibitors ICRF-193 and VP-16. Purified Metnase greatly enhanced Topo IIα decatenation of kinetoplast DNA to relaxed circular forms. Nuclear extracts containing Metnase decatenated kDNA more rapidly than those without Metnase, and neutralizing anti-sera against Metnase reversed that enhancement of decatenation. Metnase automethylates at K485, and the presence of a methyl donor blocked the enhancement of Topo IIα decatenation by Metnase, implying an internal regulatory inhibition. Thus, Metnase enhances Topo IIα decatenation, and this activity is repressed by automethylation. These results suggest that cancer cells could subvert Metnase to mediate clinically relevant resistance to Topo IIα inhibitors.

DNA replication produces tangled, or catenated, chromatids, that must be decatenated prior to mitosis or catastrophic genomic damage will occur. Topoisomerase IIα (Topo IIα) is the primary decatenating enzyme. Cells monitor catenation status and activate decatenation checkpoints when decatenation is incomplete, which occurs when Topo IIα is inhibited by chemotherapy agents such as the anthracyclines and epididophyllotoxins. We recently demonstrated that the DNA repair component Metnase (also called SETMAR) enhances Topo IIα-mediated decatenation, and hypothesized that Metnase could mediate resistance to Topo IIα inhibitors. Here we show that Metnase interacts with Topo IIα in breast cancer cells, and that reducing Metnase expression significantly increases metaphase decatenation checkpoint arrest. Repression of Metnase sensitizes breast cancer cells to Topo IIα inhibitors, and directly blocks the inhibitory effect of the anthracycline adriamycin on Topo IIα-mediated decatenation in vitro. Thus, Metnase may mediate resistance to Topo IIα inhibitors, and could be a biomarker for clinical sensitivity to anthracyclines. Metnase could also become an important target for combination chemotherapy with current Topo IIα inhibitors, specifically in anthracycline-resistant breast cancer.

Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNAdamaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, -C, -E, -F, and -G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here Fanconi anemia (FANC) 1 is an autosomal recessive disorder characterized by chromosomal instability and defective DNA repair, and FANC-deficient cells exhibit extreme sensitivity toward oxygen and DNA-cross-linking agents such as diepoxybutane and mitomycin C (1-3). The gene products of eight complementation groups of FANC have been identified and cloned (FANCA, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, and FANCL) (1-3). Mutations in any of the eight different genes lead to FANC disease, a degree of genetic heterogeneity comparable with that of other DNA repair disorders, suggesting that each group represents a distinct protein.FANCA and FANCG proteins are part of a large nuclear protein complex required for their function, and the disruption of this complex results in the specific cellular and clinical phenotype common to most FANC complementation groups (4). FANCA gene encodes a 162-kDa phosphoprotein and its phosphorylation correlated with FANCA/FANCC protein accumulation in the nucleus (5). FANCA mutant cells isolated from a FANC patient were defective in their phosphorylation and failed to bind to FANCC. Furthermore, a mutant FANCA protein failed to complement the mitomycin C (MMC) sensitivity of FANCAϪ/Ϫ cells, suggesting that FANCA phosphorylation may be involved in FANCC interaction, nuclear localization of FANCA, or its function in cross-link repair. FANCG gene encodes a 65-kDa protein that has been identified as human XRCC9. XRCC9 (FANCG) complements the Chinese hamster ovary mutant UV-40 cell line that is hypersensitive to UV, ionizing radiation, simple alkylating agents, and DNA-crosslinking agents (6, 7). The mutant cells also show a high level of spontaneous chromosomal aberrations that can be fully corrected by introduction of XRCC9 cDNA transformants (7). The possibility of the involvement of FANC proteins in DNA repair was strengthened by recent findings on the interaction of FANCD1 with BRCA1 following DNA damage (8). FANCD1 is identical to BRCA2 gene and is unique among FANC genes in that it is essential for the formation of Rad51 foci in response to ionizing radiation (9), suggesting that it may be involved in homologous recombination-mediated strand break repair.Cells lacking FANC gene showed a hypersensitive phenotype following H 2 O 2 treatment, suggesting a role for FANC proteins in redox signaling and repair of oxidative DNA damages (Refs. 10 -13 and data not shown). Interaction between FANCA and FANCG was well established by coimmunoprecipitation, cellular localization, and yeast two-hybrid analysis (4, 14 -18). Although the detailed functions of FANC proteins have yet to be determined, there is a growi...

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. While there has been a distinguished effort in identifying effectors of Chk1 activity, there are still mechanisms of its activities that are yet to be identified. Metnase/SETMAR is a SET and transposase domain protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild type (wt) Metnase associates with chromatin near an artificially induced DSB in an engineered cell system. However, an S495A Metnase mutant, which could not be phosphorylated by Chk1, had a defect in its DSB chromatin association. The S495A mutant also failed to support repair of an induced DSB when compared with wild type (wt) Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared to wt Metnase. Thus, S495 phosphorylation of Metnase differentiates between its two main functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart.

Zhu, W-G., Seno, J. D., Beck, B. D. and Dynlacht, J. R. Translocation of MRE11 from the Nucleus to the Cytoplasm as a Mechanism of Radiosensitization by Heat. Radiat. Res. 156, 95-102 (2001).Hyperthermia sensitizes mammalian cells to ionizing radiation, presumably by inhibiting the repair of radiation-induced double-strand breaks (DSBs). However, the mechanism by which heat inhibits DSB repair is unclear. The nuclear protein MRE11 is a component of a multi-protein complex involved in nonhomologous end joining (NHEJ) of radiation-induced DSBs. Using one-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blotting, we found that MRE11 is translocated from the nucleus to the cytoplasm when human U-1 melanoma or HeLa cells are heated for 15 min at 45.5 degrees C or when cells are heated after irradiation with 12 Gy of X rays. No such translocation is observed in unheated irradiated cells. The kinetics of migration of MRE11 to the cytoplasm was dependent upon whether the heated cells were irradiated, while the magnitude of redistribution of MRE11 was dependent upon post-treatment incubation time at 37 degrees C. Cytoplasmic MRE11 content reached a maximum 2-4 h after heating; the increase was not due to new protein synthesis. Partial recovery of nuclear MRE11 content was observed when heated cells or heated irradiated cells were incubated for up to 7 h at 37 degrees C after treatment. Western blotting results showing translocation of MRE11 from the nucleus to the cytoplasm after heating and irradiation were confirmed using confocal microscopy and immunofluorescence staining of fixed cells. Our data suggest that radiosensitization by heat may be caused, at least in part, by translocation of the DNA repair protein MRE11 from the nucleus to the cytoplasm.

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.