Brian D. Beck

Human Pso4 Is a Metnase (SETMAR)-binding Partner That Regulates Metnase Function in DNA Repair

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.

Translocation of MRE11 from the nucleus to the cytoplasm as a mechanism of radiosensitization by heat.

Abstract 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°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°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°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.

The SET and transposase domain protein Metnase enhances chromosome decatenation: regulation by automethylation

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.

Metnase Mediates Resistance to Topoisomerase II Inhibitors in Breast Cancer Cells

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.

Biochemical characterization of Metnase's endonuclease activity and its role in NHEJ repair

Metnase (SETMAR) is a SET-transposase fusion protein that promotes nonhomologous 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 Metnases endonuclease activity in promoting the joining of noncompatible ends.

Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells.

Abstract Beck, B. D. and Dynlacht, J. R. Heat-Induced Aggregation of XRCC5 (Ku80) in Nontolerant and Thermotolerant Cells. Radiat. Res. 156, 767–774 (2001). 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°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°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.

Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart.

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart 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 DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two 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.

The non-homologous end-joining pathway is not involved in the radiosensitization of mammalian cells by heat shock.

A synergistic increase in cell killing is observed when a heat‐shock is administered prior to, during, or immediately after exposure to ionizing radiation (IR). This phenomenon, known as heat‐radiosensitization, is believed to be mediated by inhibition of repair of radiation‐induced double strand breaks (DSB) when cells are exposed to temperatures above 42°C. However, the mechanism by which heat inhibits DSB repair is unclear. The bulk of radiation‐induced DSBs are repaired via the non‐homologous end‐joining pathway (NHEJ). Several reports indicate that the Ku70 and Ku80 subunits of the mammalian DNA‐dependent protein kinase (DNA‐PK), a complex involved in NHEJ, appear to be susceptible to a heat‐induced loss of DNA‐binding activity, with Ku80 representing the heat‐sensitive component. Since the heat‐induced loss and subsequent recovery of Ku–DNA binding activity correlates well with heat‐radiosensitization, a role for Ku80 and NHEJ in heat‐radiosensitization has been proposed. However, direct evidence implicating Ku80 (and NHEJ) in heat‐radiosensitization has been indeterminate. In this study, we demonstrate that equitoxic heat treatments at 42.5–45.5°C induce a similar amount of aggregation of Ku80 in human U‐1 melanoma cells. These data suggest that the time–temperature‐dependent relationship between heat lethality and Ku80 aggregation are similar. However, the aggregation/disaggregation of Ku80 and its transient or permanent inactivation is unrelated to heat‐radiosensitization. When survival curves were obtained for irradiated or irradiated and heated Ku80−/− mouse embryo fibroblasts (MEFs) and compared with survival curves obtained for wild‐type (WT) cells, we found that heat‐radiosensitization was not reduced in the Ku80−/− cells, but actually increased. Thus, our findings indicate that Ku80 is not essential for heat‐radiosensitization. Non‐involvement of Ku‐dependent or Ku‐independent NHEJ pathways in heat‐radiosensitization was confirmed by comparing clonogenic survival between DNA ligase IV‐defective and WT human cells. Our data therefore implicate homologous recombination in inhibition of repair of radiation‐induced DSBs and as a target for heat‐radiosensitization. J. Cell. Physiol. 196: 557–564, 2003.

XPB and XPD between Transcription and DNA Repair

Xeroderma pigmentosum group B and D genes (XPB and XPD respectively) are components of the transcription factor IIH (TFIIH), a nine-subunit complex involved in transcription initiation by RNA polymerase II (pol II). Five of these (XPB, p62, p52, p44 and p34) form a tight core subcomplex, while XPD is less tightly associated with the core and mediates the binding of the CAK subcomplex, containing the remaining three subunits, cyclin H, cdk7 and MAT1.1,2 The TFIIH complex also plays a key role in nucleotide excision repair (NER) by opening duplex DNA at the damage site. Both XPB and XPD possess DNA helicase activities, though XPB functions in a 3′ → 5′ fashion,3 while XPD catalyzes unwinding of duplex DNA in the opposite direction.2 The DNA-dependent helicase activity of XPB and XPD is central to the function of TFIIH in both transcription initiation and NER.

Fanconi Anemia D2 Protein Is an Apoptotic Target Mediated by Caspases

FANCD2, a key factor in the FANC‐BRCA1 pathway is monoubiquitinated and targeted to discrete nuclear foci following DNA damage. Since monoubiquitination of FANCD2 is a crucial indicator for cellular response to DNA damage, we monitored the fate of FANCD2 and its monoubiquitination following DNA damage. Disappearance of FANCD2 protein was induced following DNA damage in a dose‐dependent manner, which correlated with degradation of BRCA1 and poly‐ADP ribose polymerase (PARP), known targets for caspase‐mediated apoptosis. Disappearance of FANCD2 was not affected by a proteasome inhibitor but was blocked by a caspase inhibitor. DNA damage‐induced disappearance of FANCD2 was also observed in cells lacking FANCA, suggesting that disappearance of FANCD2 does not depend on FANC‐BRCA1 pathway and FANCD2 monoubiquitination. In keeping with this, cells treated with TNF‐α, an apoptotic stimulus without causing any DNA damage, also induced disappearance of FANCD2 without monoubiquitination. Together, our data suggest that FANCD2 is a target for caspase‐mediated apoptotic pathway, which may be an early indicator for apoptotic cell death. J. Cell. Biochem. 112: 2383–2391, 2011.