Metnase promotes restart and repair of stalled and collapsed replication forks

Abstract

Metnase is a human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes non-homologous end-joining (NHEJ), and knockdown causes mild hypersensitivity to ionizing radiation. Metnase also promotes plasmid and viral DNA integration, and topoisomerase IIα (TopoIIα)-dependent chromosome decatenation. NHEJ factors have been implicated in the replication stress response, and TopoIIα has been proposed to relax positive supercoils in front of replication forks. Here we show that Metnase promotes cell proliferation, but it does not alter cell cycle distributions, or replication fork progression. However, Metnase knockdown sensitizes cells to replication stress and confers a marked defect in restart of stalled replication forks. Metnase promotes resolution of phosphorylated histone H2AX, a marker of DNA double-strand breaks at collapsed forks, and it co-immunoprecipitates with PCNA and RAD9, a member of the PCNA-like RAD9-HUS1-RAD1 intra-S checkpoint complex. Metnase also promotes TopoIIα-mediated relaxation of positively supercoiled DNA. Metnase is not required for RAD51 focus formation after replication stress, but Metnase knockdown cells show increased RAD51 foci in the presence or absence of replication stress. These results establish Metnase as a key factor that promotes restart of stalled replication forks, and implicate Metnase in the repair of collapsed forks.

Figure 1.

Metnase promotes cell proliferation. (A) Cell growth was monitored in HEK-293 cells transfected with shGFP (control) or shMetnase vectors. (B) Cell growth was monitored in HEK-293-T cells, which do not normally express Metnase, transfected with the pCAPP-Metnase expression vector or empty pCAPP. Plotted are averages (±SD) of two to three determinations per time-point. *P < 0.05, **P < 0.01. Metnase expression is shown in representative western blots with β-actin loading control (insets). Cell growth was measured by harvesting cells at indicated times and counting cells with a Coulter counter.

Figure 2.

Metnase promotes cell survival after DNA replication stress. (A) Average percent cell survival (± SD) after HU, CPT or UV-B treatments measured as relative plating efficiency for HT1080 or HEK-293 cells stably transfected with control or shRNA-Metnase vectors. Data are from two to three independent experiments per condition; *P = 0.0127, **P ≤ 0.01. (B) Average growth rates (±SD) of control HEK-293 and sh-Metnase knockdown cells, and control HEK-293T or Metnase overexpression cells in medium containing 0.1 mM HU; data are from two to three independent experiments per cell line. (C) HEK-293 control or Metnase knockdown cells were treated with 5 mM HU for 6 h and the percentages of cells expressing annexin V or incorporating propidium iodide were determined by flow cytometry. Values are averages (±SD) from three independent experiments.

Figure 3.

Metnase promotes DNA replication after release from replication stress. (A) Log phase HEK-293 cells expressing normal or low levels of Metnase were incubated with 10 µM BrdU for 30 min and average percentages (±SD) of BrdU⁺ cells are shown for two determinations per strain. (B) HEK-293 control and Metnase knockdown cells were treated with 5 mM HU for 3 h and released into medium with 10 µM BrdU. Average fold increases (±SD) in the percentage of BrdU⁺ cells relative to untreated cells (no HU, no BrdU) are plotted for three independent experiments per cell line. *P = 0.042, **P = 0.0047. (C) BrdU incorporation after HU release from HEK-293T control and Metnase overexpression cells as in panel B, except cells were treated with HU for 18 h; *P ≤ 0.03.

Figure 4.

Metnase promotes replication fork restart. (A) IdU and CldU labeling scheme is shown above representative confocal microscope images of DNA fibers, with IdU stained red and CldU stained green. (B) Quantification of fiber types. At least 150 fibers were scored per treatment, per cell line for each of three experiments; **P ≤ 0.0014. (C) Fiber lengths were measured by using LSM 510 Image Browser software. Plotted are averages (±SD) of triplicate experiments in which 150–500 fibers were scored per treatment, per experiment. nd, none detected.

Figure 5.

Metnase promotes resolution of replication stress-induced γ-H2AX. (A) Representative confocal microscope images of HEK-293 and HEK-293T cells over- or under-expressing Metnase were treated with 10 mM HU for 18 h and released into growth medium for 24 h. Aliquots of cells were removed at indicated times, cytospun, stained with DAPI (blue) and antibodies to γH2AX (green) and imaged by confocal microscopy. (B, C) Percentage of γ-H2AX positive cells among total DAPI stained cells. An average of >190 cells were counted per slide, 10 slides per experiment. Values are averages (±SD); **P ≤ 0.0055.

Figure 6.

Metnase interacts with PCNA and RAD9, but not RPA32. (A) Reciprocal co-immunoprecipitation of V5-tagged Metnase and native PCNA from cells treated with 5 mM HU for 18 h, tested immediately or 30 min after release from HU, or untreated. Input represents 0.5% of immunoprecipitation. Results are representative of at least three independent experiments. (B and C) Co-immunoprecipitation of V5-tagged Metnase with native RAD9 and native RPA as in panel A, except HU treated cells were only tested immediately after treatment.

Figure 7.

Metnase interacts with TopoIIα and stimulates relaxation of positive supercoils. (A) Predominantly positively-supercoiled plasmid DNA samples were treated with TopoIIα (2 U) with or without Metnase (180 ng) for indicated times, and topological forms were detected on ethidium bromide stained agarose gels. (B) Gel images were scanned and the percentage of positively-supercoiled DNA remaining at each time point was quantitated. Values are averages (±SD) of two determinations per condition, normalized to 100% at t = 0; **P = 0.007. (C) Co-immunoprecipitation of V5-tagged Metnase and native TopoIIα; data presented as in Figure 6B.

Figure 8.

Potential roles of Metnase in the replication stress response. See text for details.