MUS81-EME2 promotes replication fork restart

Abstract

Replication forks frequently stall at regions of the genome that are difficult to replicate or contain lesions that cause replication blockage. An important mechanism for the restart of a stalled fork involves endonucleolytic cleavage that can lead to fork restoration and replication progression. Here, we show that the structure-selective endonuclease MUS81-EME2 is responsible for fork cleavage and restart in human cells. The MUS81-EME2 protein, whose actions are restricted to S phase, is also responsible for telomere maintenance in telomerase-negative ALT (Alternative Lengthening of Telomeres) cells. In contrast, the G2/M functions of MUS81, such as the cleavage of recombination intermediates and fragile site expression, are promoted by MUS81-EME1. These results define distinct and temporal roles for MUS81-EME1 and MUS81-EME2 in the maintenance of genome stability.

Graphical abstract

Figure 1

S Phase MUS81-EME2 Is Not Required for SCE Formation (A) Schematic representation of BAC-MUS81_FLAP. (B) HeLa Kyoto cells expressing MUS81_FLAP were synchronized at G1/S using a double thymidine block. Six hours after release, the cycling cells were supplemented with nocodazole to promote G2/M phase arrest. Samples were collected at 0, 3, 6, 9, and 12 hr as indicated, and cell-free extracts were prepared. MUS81_FLAP was immunoprecipitated from the extracts and the presence of each indicated protein was determined by western blotting. (C) Cell-cycle progression of the cells used in (B) as determined by FACS analysis. (D) Representative images of metaphase spreads prepared from BLM-deficient cells (GM08505) transfected with siRNAs against Luciferase (siControl), EME2, EME1, or MUS81. Scale bar, 10 μm. (E) Quantification of SCE formation in cells treated with the indicated siRNAs, as shown in (D). For each condition, 32 metaphases were analyzed and each data point represents the number of SCEs per 100 chromosomes per metaphase spread; p values were determined using the two-tailed unpaired t test with Welch correction.

Figure 2

MUS81-EME2 Promotes the Breakage and Restart of Stalled Replication Forks (A) HeLa cells depleted for MUS81, EME1, or EME2 were treated with the indicated concentrations of HU for 24 hr and the DNA was analyzed for breaks by PFGE. (B) Quantification of HU-induced DSB formation, as determined in (A), expressed as the ratio of broken to intact DNA. Data are presented as a mean of three experiments (± SEM). (C) HeLa cells transfected with the indicated siRNAs for 48 hr before addition of HU (2 mM for 24 hr) were analyzed by fiber analyses for replication fork progression. Representative images of iododeoxyuridine (IdU)-labeled (green) and chlorodeoxyuridine (CldU)-labeled (red) DNA fibers are shown. (D) Quantification of the fiber analyses indicated in (C) ± SEM. Statistical significance was calculated using Fisher’s exact test; 300 fibers were scored for each condition. See also Figure S1.

Figure 3

MUS81-EME2 Is Required for Genome Stability (A) Representative images of chromosomal aberrations observed in metaphase spreads prepared from MUS81- or EME2-depleted RPE-1 hTERT cells following HU treatment. Images were categorized as breaks, radials, dicentrics, or acentrics, and quantified as described in (B) and Table S1. (B) Quantification of the frequency of chromosomal breaks and fragments in siRNA-depleted RPE-1 hTERT cells with or without HU (1 mM, 24 hr) treatment. Data are presented as the mean of three experiments (± SEM). Statistical significance was calculated using Student’s t test. 50 metaphase spreads were scored for each condition. (C) Quantification of anaphase bridge formation in siRNA-depleted treated cells following treatment with either APH or HU (± SEM). Cells were transfected with the indicated siRNAs for 48 hr before treatment with APH (150 nM for 16 hr) or HU (1 mM for 24 hr). HU-treated cells were incubated in fresh media for 24 hr before staining. Statistical significance was calculated using Fisher’s exact test (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001); 300 cells were scored for each condition. (D) Representative image of a metaphase spread of RPE1-hTERT cells transfected with control siRNA and treated with 150 nM APH for 16 hr. Scale bar, 10 μm. The arrow indicates a typical break as quantified in (E). (E) Quantification of breaks in metaphase spreads in the indicated siRNA-depleted cells following treatment with 150 nM APH for 16 hr (± SEM). Statistical significance was calculated using Student’s t test; 50 metaphases were scored for each condition.

Figure 4

MUS81-EME2 Is Required for Telomere Maintenance in ALT Cells (A) Representative Q-FISH images of metaphase spreads from U2OS cells depleted for the indicated proteins. Telomeric repeats are indicated in red. The arrows point to a normal chromosome (A) or to chromosomes with telomere-free ends (B and C). (B) Quantification of telomere-free chromosome ends in the depleted cells (± SEM), as in (A). (C) Representative CO-FISH images of metaphase spreads from U2OS cells depleted for the indicated proteins. T-SCEs were detected using telomeric G-strand (red) and C-strand (green) PNA probes. Arrows point to chromosomes without T-SCEs (A) and to chromosomes with telomeric exchange signals (yellow, B and C). (D) Quantification of chromosome ends displaying T-SCEs in the depleted U2OS cells (± SEM), as in (C). In (B) and (D), statistical significance was calculated using Pearson’s chi-square test; n indicates the number of chromosome ends analyzed. See also Figures S2–S4.