Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of licensing factor Cdt1 in the G1 phase

Abstract

Cdt1 is rapidly degraded by CRL4^Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ∼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ∼30 min in NER-deficient XP-A cells, but was degraded within ∼60 min. The delayed degradation was also dependent on PCNA and CRL4^Cdt2. The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4^Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.

Keywords: CRL4Cdt2; Cdt1; Mismatch repair (MMR); PCNA; UV.

Figure 1.

Cdt1 degradation in XP-A cells after UV irradiation is delayed and it is also dependent on PCNA and CRL4^Cdt2. (A) Normal fibroblasts (NF) and XP-A cells were irradiated with UV at 5 J/m² and collected at the indicated time-points for Western blotting. RCC1 was used as a loading control. (B) Cells grown on coverslips were irradiated as in (A) and fixed for staining with antibodies for Cdt1 and Cyclin A (CyA). Cells positive for Cdt1 (+) or Cyclin A (+) were counted at the indicated time-points and their frequency was plotted (%) for both NF and XP-A cells. (C) XP-A cells and XP-A cells complemented with the wild-type XPA gene (wtXPA) were UV-irradiated (5 J/m²) and collected at the indicated time-points for Western blotting (left panel). Comparison of the time-course of Cdt1 degradation in XP-A, XP-A(wtXOA), and NF after UV irradiation (right panel; means and standard deviation of 3 independent experiments are shown.) (D) The delayed Cdt1 degradtion in XP-A cells is also dependent on ubiquitin-proteasome pathway mediated by CRL4^Cdt2. NF or XP-A cells were incubated with MG132, 10 min later irradiated with UV at 5 J/m² and collected at the indicated time-point (min) for Western blotting (left upper panel). Cultures of cells were transfected with the indicated siRNAs twice and incubated for a total of 3 d. Cells were irradiated with UV at 5 J/m² and collected at the indicated time-points (min) for Western blotting. Cdt1 protein levels were measured and its relative amounts, normalized to RCC1, were shown. siRNA for luciferase was used as a control (siLuc). Asterisks indicate a non-specific band.

Figure 2.

PCNA, Cdt2, and Cdt1 accumulate at the UV-irradiated sites in XP-A cells, but later than in normal fibroblasts. (A) Asynchronously growing normal fibroblasts (NF) and XP-A cells were covered with a membrane with 5-μm pores, irradiated locally with UV (100 J/m²; micropore assay), and incubated for the indicated times. The cells were fixed and stained with antibodies for CPD and PCNA. Bar, 10 μm. The white triangles in UV (-) indicate S phase cells (see text). (B) PCNA intensity was measured using ImageJ at the CPD spots as shown in (A) (> 25 spots for each time-point) and mean values (arbitrary) were plotted. (C) Asynchronously growing NF, XP-A, and XP-A(wtXPA) cells were treated as in (A), incubated for 30 min, and stained with antibodies for CPD and PCNA. (D) The intensity of PCNA at the CPD sites in (C) was measured using ImageJ (30 spots/cell line) and mean values (arbitrary) are shown. (E) Asynchronously growing cells were treated as in (A) and stained with antibodies for CPD and Cdt1. To visualize Cdt1 proteins, MG132 was added to the medium 10 min before UV irradiation. Bar, 5 μm. Cells were examined for Cdt1 staining at CPD sites (> 100 sites for each time-point) and the frequency of Cdt1-positive sites is shown (%) (left panel). Cdt1 intensity of cells was also measured and mean values are shown (arbitrary) (right panel).

Figure 3.

Msh2 and Msh6 proteins were recruited to the UV-irradiated sites in XP-A cells. (A) Cells synchronized in the G1 phase were locally irradiated (+) or not (−) through a membrane with 5-μm pores. Cells were incubated for 1 h, pre-extracted, and fixed for staining with CPD and Msh2 or Msh6 antibodies. (B) Cells were examined for Msh2 or Msh6 staining at CPD sites. Frequency of CPD sites that were positive for Msh2 or Msh6 are shown (%) (> 100 CPD sites were examined). (C) XP-A cells treated as in (A) were co-stained with Msh2 and PCNA. 92.5% of CPD sites were positive for Msh2 (62/67 CPD sites). (D) Cells synchronized in the G1 phase were locally irradiated with UV (+) and fixed for staining with antibodies for CPD and Msh2 at the indicated time-points. (−): not irradiated with UV. Cells were examined for CPD and Msh2, and the frequency of cells co-stained with CPD and Msh2 was plotted. Bar, 5 μm.

Figure 4.

MMR is involved in the Cdt1 degradation in XP-A cells. (A) XP-A cells were transfected twice with the siRNAs for Ape1, Msh2, Msh6, or luciferase (siLuc) as a control. Three days later, half of the cell cultures were irradiated with UV at 5 J/m² and incubated for the indicated period of time. Cells were collected at the indicated time-points for Western blotting with antibodies for Cdt1, Msh2, Msh6, Ape1, and RCC1. Cdt1 protein levels were measured and its relative amounts were shown. (B) XP-A cells were treated as in (A) and fixed and stained with antibodies for Cdt1 and Cyclin A (CyA). Cells were examined for Cdt1 staining and the frequency of Cdt1-positive cells is shown (%). Bar, 10 μm.

Figure 5.

MMR is involved in Cdt1 degradation in normal cells. (A) Normal fibroblasts (NF) were transfected with the indicated siRNAs and 3 d later, UV irradiated (25 J/m²) and collected at the indicated time-points. Cdt1 protein levels were normalized with Mcm6 protein and mean values and standard deviation of 3 independent experiments are shown. (B) U2OS cells were treated as in (A) and analyzed. (means and standard deviation of 3 independent experiments are shown). (C) Interaction of Cdt2–3xFLAG with MMR proteins. Cdt2–3xFLAG–expressing stable U2OS cells were isolated, and examined for expression levels of Cdt2–3xFLAG (3xFLAG) and endogenous Cdt2 (end.) by Western blotting with Cdt2 antibody. U2OS cells and Cdt2–3xFLAG–expressing U2OS cells were treated with MG132 for 10 min and UV irradiated (25 J/m²) (+) or not (−). After incubation for 30 min, the cells were collected, fixed with paraformaldehyde, and immunoprecipitated with anti-FLAG as described in the Materials and Methods. Precipitates were examined for the indicated proteins. The band intensities for Msh2, Mas6 and PCNA were measured and normalized to UV(−). Means and standard deviation of 3 independent experiments are shown for Mas2 and Mas6. For PCNA, the mean of 2 independent assays is shown. Asterisk indicates Msh2 bands remaining after re-blotting with anti Cdt2 antibody.

Figure 6.

Cdt1 degradation is important for DNA repair synthesis. (A) HeLa cells were transfected with Cdt1(1–101)-Cy-9myc or 9myc only. The next day, cells were locally irradiated with UV, incubated in the presence of EdU for 30 min, and fixed for staining with antibodies for myc and Alexa Fluor 488 azide to detect EdU as supplied by the manufacturer. The open arrow indicates Cdt1(1–101)-Cy-9myc expressing cells with low EdU staining, while the filled arrows indicate 9myc-only–expressing cells with EdU spot signals. (B) EdU spot-positive cells in (A) were examined for expression of Cdt1(1–101)-Cy-9myc (Cdt1-Cy) or 9myc-only, and the frequency of cells not expressing (blue) or expressing (red) (%) is shown for each transfected cell culture. The mean of 2 independent assays is shown. (C) HeLa cells were treated as described in A, and stained for EdU and CPD. Open arrows indicate CPD sites with low or negative EdU signals. (D) CPD spot-positive cells in (C) were examined for EdU incorporation, and the frequency of EdU-positive (+; green) or -negative or low (-; white) cells (%) are shown for each transfected cell culture. The mean of 2 independent assays is shown.