The ERCC1/XPF endonuclease is required for efficient single-strand annealing and gene conversion in mammalian cells

Cell lines

The cell lines used are UV4DR7, ERCC1.17, ERCC1.21 and PEF7. The UV4DR7 cell line was created following electroporation of the DRneo vector and isolation of individual clones as described earlier (20). To obtain an ERCC1-complemented UV4DR7 cell line, the ERCC1 gene was cloned into the PEF6-V5-His-Topo vector according to the manufacturer's protocol (Invitrogen). The ERCC1 cDNA was amplified in a two-step RT-PCR with RNA extracted from SPD8 cells using primers; ERCC1-f 5′-CAG ATG GAC CTT GGG AAA GAC-3′ and ERCC1-r 5′-TTA TGA CGC TGT AGC CTC AGC-3′. A total of 5 × 10⁶ cells/ml were electroporated with 30 μg of pEF6-V5-His-ERCC1 or empty control vector (1500 μF capacity, 250 V for 30 s). Blasticidin (5 µg/ml) was added 48 h following electroporation and individual blasticidin resistant colonies isolated and expanded. All cell lines were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum, penicillin (60 µg/ml), streptomycin (100 µg/ml) at 37°C containing 5% CO₂, in a humidified incubator. The medium was supplemented with hygromycin (0.05 mM) in order to maintain the DRneo vector, and Blasticidin (3 µg/ml) to maintain the PEf6-V5-His-ERCC1 and PEF6-V5-His-Topo vector-containing cell lines.

Southern blotting

Genomic DNA was isolated from the UV4DR7 cell line, phenol–chloroform treated and digested with restriction endonucleases. Ten microgram of purified DNA was digested HindIII, HindIII + SacI, HindIII + KpnI and/or HindIII + NcoI was used. To determine that only one integrated copy of DRneo was present in the genome. The HindIII + XhoI was used to determine the size of the DRneo substrate in G418^R clones. All DNA was separated by gel electrophoresis and Southern blotting was carried out as previously described using the S2neo fragment as probe (20).

UV survival assay

Five hundred cells from each cell line were seeded over night to be treated with different doses of UV (0, 0.2, 0.5, 1, 2, 5 and 10 J/m²) on the following day. After that they were incubated for 7–14 days in a humidified 5% CO₂/37C incubator and then the colonies were fixed and stained with 0.4% methylene blue in methanol and individual colonies (greater than 30 cells) counted.

Recombination assay

To avoid contamination with spontaneous G418 resistant clones in the DRneo recombination assay, 10³ cells from each cell lines were separately expanded to confluencency on about 10 different Petri dishes for each cell line (Ø 100 mm). The spontaneous recombination frequency was determined for each cell population by selection of 2 × 10⁵ cells/plate (Ø 100 mm) seeded in G418 (100 μg/ml). Only those cell populations that did not retrieve viable G418^R colonies were used for further recombination assays, as they have low background frequency of spontaneous G418^R cells.

In the recombination assay, 1.5 × 10⁶ cells of each cell line (UV4DR7, ERCC1.17, ERCC1.21 and PEF7) were seeded over night, and transiently transfected with or without pCMV3xnls-I-SceI expression vector (100 ng) according to manufacturer's protocol (Lipofectamine2000™, Invitrogen, UK, final DNA concentration 10 µg/ml) to create DSBs. After 5 h of incubation in the humidified 5% CO₂/37°C incubator, cells were changed to complete medium. Twenty-four hours after transfection, the cells were trypsinized and counted. For recombination frequency, 2 × 10⁵ cells/plate (Ø 100 mm) were seeded and G418 (100 μg/ml) and/or hygromycin (0.5 mg/ml) were added for selection to both treated and untreated plates. A total of 500 cells/plate were seeded to determine the cloning efficiency. Plates were incubated for 7 or 10 days for cloning and selection, respectively, after which they were fixed and stained with 0.4% methylene blue in methanol.

For synchronization of cells in G1 phase of the cell cycle, we plated 1.5 × 10⁶ ERCC1.17 and 2.5 × 10⁶ UV4DR7 cells for 48 h to reach contact inhibition, or 2 × 10⁶ ERCC1.17 and UV4DR7 cells were grown for 48 h in serum-free media. Cells were then transiently transfected with either pCMV3xnls-I-SceI or pEGFP-C2 vectors and then followed the above protocol for cloning and selection, or treated with inhibitors throughout the transfection with 2 mM caffeine, 10 µM KU55933, 10 µM NU7026, 25 µM Roscovitine or 500 nM CEP-3891. All experiments were repeated independently at least three times.

FACS analysis

To determine lipofectamin transfection rates, cells were transiently transfected with a reporter vector, pEGFP-C2 Vector (Invitrogen), using the manufacturer's protocol (Invitrogen) and expression of EGFP was analysed by FACS Sort Vantage (Becton Dickinson, UK).

For cell cycle analysis, arrested cells were harvested, washed twice in PBS and fixed in 70% cold ethanol followed by treatment with 1 mg/ml RnaseA and staining with 100 mg/ml propidium iodide. The proportion of arrested cells in each phase of cell cycle was then determined by FACS Sort Vantage.

Western blotting

A total of 2 × 10⁶ cells was plated onto 100 mm dishes and grown for 24 h. Cells were then lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 0.1 mM PMSF in PBS) in the presence of 1× protease and phosphatase inhibitor cocktails (Sigma, UK). An aliquot of 50 µg total protein was then run on a 10% SDS–PAGE gel and transferred to Hybond C membrane (Amersham Pharmacia, UK) using a semi-dry transfer cell (Bio-Rad). This membrane was blocked in 5% milk for 1 h and immunoblotted with mouse monoclonal antibody ERCC1 vAb-2 (clone 8F1; Labvision), diluted 1:200 in 5% milk in PBS-0.05% Tween and rabbit anti-actin (Sigma) antibody diluted 1:2000 in 5% milk in PBS-0.05% Tween overnight. Anti-mouse peroxidase conjugate (Sigma 1:2000) and anti-rabbit peroxidase conjugate (Cell Signaling 1:1000) were used, respectively, as secondary antibodies and immunoreactive protein was visualized using ECL reagents (Amersham Pharmacia) following manufacturer's instructions.

ERCC1 is involved in SSA and gene conversion

Here, we studied the role of ERCC1 using a recombination reporter construct, DRneo (21), which contains a hygromycin (Hyg) resistance gene and two non-functional copies of the neo^R gene. After introduction of a DSB, these non-functional copies revert to a functional neo^R gene through either SSA, sister chromatid exchange (SCE) or gene conversion, conferring resistance to geneticin (G418) (Figure 1A). We first stably integrated the DRneo reporter construct into the genome of the UV4 hamster cell line that is deficient in ERCC1 (1), and isolated individual hygromycin-resistant clones. The presence and correct integration of a single DRneo copy into the UV4DR7 clone was confirmed using Southern blotting with S2neo as probe (Figure 1B). HindIII cleavage resulted in a single 6.5 kb band, showing that the DRneo construct is present in one copy (Figure 1B). HindIII and KpnI or SacI cleavage gave the same 6.5 kb band, which implies that KpnI and SacI cleave outside the 6.5 kb region. HindIII and NcoI cleavage produced a 4.7 kb and a 1.4 kb band, showing that the internal structure of DRneo is preserved in the UV4DR7 clone.

The pCMV3xnlsI-SceI vector was transiently transfected into UV4DR7 cells to express the I-SceI endonuclease to induce a DSB in the DRneo substrate. We performed a fluctuation assay to determine the extent of recombination induced and found that a DSB induces HR 10³-fold (Figure 1C). This shows that ERCC1-defective cells have no overall defect in HR.

We transfected the UV4DR7 cell line with the pEF6-V5-His vector containing the hamster ERCC1 gene, or with the empty control vector. Individual clones were isolated and the level of ERCC1 expression quantified by western blotting, using extracts from hamster AA8 and human HeLa cells as control (Figure 2A). The ERCC1.17 and ERCC1.21 clones expressed ERCC1 to the same extent as wild-type hamster AA8 cells and fully complemented the sensitivity of ERCC1-defective cells to UV treatments (Figure 2B). In contrast, the clone PEF7 containing the empty control vector was still sensitive to UV as a result of the continued defect in ERCC1. The reason for the slight UV sensitivity difference to UV4DR7 is likely explained by that PEF7 the fact is an isolated clone from UV4DR7.

We investigated HR in the UV4DR7, ERCC1.17, ERCC1.21 and PEF.7 cell lines by transiently transfecting the cells with pCMV3xnlsI-SceI vector to create a DSB in the S2neo fragment of the DRneo reporter construct. Recombination of DRneo to a neo^R gene can occur through either gene conversion, SSA or SCE (Figure 1A). If the neo^R gene is restored by gene conversion, the hyg^R gene in between the repeats will be retained, while it will be lost following SSA or SCE (Figure 1A). We selected recombinant clones with G418 or G418 plus hygromycin (G418 + Hyg) to determine the frequency of the different recombination events.

If ERCC1 is exclusively involved in SSA, it would be expected that the levels of G418 resistant, but not G418 + Hyg resistant, clones would be lower in UV4DR7 and PEF7 cells, as those represent the products of SSA. Surprisingly, both the levels of G418 and G418 + Hyg resistant recombinants were reduced in UV4DR7 and PEF7 cells (Figure 2C). The levels of SSA and gene conversion were reduced by 83% and 75% in ERCC1-defective cells, respectively. These data demonstrate that ERCC1 is involved not only in SSA, but also in gene conversion.

We isolated 10 G418-resistant clones and determined the recombination products using Southern blotting and compared the results to their subsequent resistance to hygromycin (Figure 2D). The presence of a 4.0 kb band in the Southern blots correlated with hygromycin resistance. Interestingly, we found a clone with both the 1.1 and 4.0 kb bands, indicating that two DRneo alleles (present after replication) reverted to neo^R genes using different mechanisms, which has been reported earlier for a similar recombination substrate (20,22). Altogether, these data show that G418 + Hyg resistance correlates with a gene conversion recombination product, which has also been shown to be the case with the same recombination reported earlier (21).

Suppressed SSA in G1-arrested cells

It is well established that HR predominates during and following replication and that NHEJ predominates in the G1 phase of the cell cycle (23,24). As SSA is generally believed to occur also in the absence of a sister chromatid, it is plausible that this could be a frequently used pathway for DSB repair in G1-arrested cells, as a complement to NHEJ. We had the DRneo substrate present in both ERCC1-defective and complemented cells, allowing us to test if SSA is specifically used for DSB repair in G1-arrested cells. We synchronized cells in the G1 phase using either serum starvation (SFM) or contact inhibition (CI) (by growing cells to confluency). We found that a 48-h SFM or CI of UV4DR7 or ERCC1.17 cells accumulated in the G1 phase (Figure 3A). The cells were subsequently transfected with the pCMV3xnlsI-SceI vector for 5 h and kept arrested for an additional 19 h, to ensure expression of the I-SceI restriction endonuclease and DSB formation while cells were still in the G1 phase, before replating for selection in G418 or G418 + Hyg. We have previously reported differential transfection efficiency between cycling and arrested cells (24). Thus, to compensate for differential effects in expression from the pCMV3xnlsI-SceI, we co-transfected cells with the same amount of pEGFP-C2 vector, expressing the green fluorescent protein (GFP) from the same CMV promoter as the one expressed in the pCMV3xnlsI-SceI vector. We found reduced GFP expression in G1-arrested cells (data not shown), in agreement with the previously published results (24). We determined the I-SceI-induced recombination frequencies in G1-arrested and cycling cells and compensated the recombination frequency of the arrested cells for the reduced transfection efficiency. In ERCC1 expressing wild-type cells, we found that both the levels of gene conversion and SSA drop in G1-arrested cells. The gene conversion level (represented by the yellow bar) dropped 2-and 4-fold in serum-starved ERCC1.17 and ERCC1.21 cells, respectively and 3-fold in both cell lines that were contact-inhibited (Figure 3B), in agreement with our earlier report that gene conversion events are reduced in G1-arrested cells (24).

Interestingly, the SSA levels in ERCC1 proficient cells (represented by blue bar minus yellow bar) dropped 5- to 6-fold in both serum-starved and contact-inhibited cells as compared to dividing control cells (statistically significant P < 0.001). Thus, the efficiency of SSA, even within perfect repeats, is reduced in G1-arrested cells.

The reduction of gene conversion and SSA was not as profound in ERCC1-defective UV4DR7 and PEF7 cells. These data suggest that ERCC1-dependent SSA and gene conversion are more efficient in cycling cells and that ERCC1-independent recombination events contribute to the recombination events that restore a functional neo^R gene in G1-arrested cells. Overall, our data suggest that ERCC1-dependent SSA and gene conversion is more efficient following replication in the S/G2 phase of the cell cycle.

SSA is independent of cell cycle checkpoint signalling

It was reported earlier that HR repair of a DSB depends on the checkpoint proteins Ataxia-Telangiectasia and Rad3-related (ATR) and Chk1 (25), and that resection of DNA ends to facilitate ATR activation and HR relies on cyclin-dependent kinases (CDK) activity (26–28). Thus, one reason for reduced SSA in G1-arrested cells could be the absence of CDK activity in these cells and thus a reduced resection at DSBs. Absence of resection is likely to prevent SSA as no ssDNA homologies would be unveiled. To test the possibility that SSA depends on cell cycle checkpoint signalling, we used various protein kinase inhibitors; Roscovitine inhibits CDK especially CDK2, which plays an essential role in initiating replication (29). Caffeine is a broad range phosphoinositide 3-kinase related kinase inhibitor, inhibiting both ATM (Ataxia-Telangiectasia Mutated) and ATR (30). KU55933 is a specific ATM inhibitor and NU7026 inhibits the DNA-dependent protein kinase (DNA-PK) required for NHEJ (31). CEP-3891 is a specific inhibitor of Chk1 (25). We treated ERCC1.17 cells with the inhibitors during transfection of the pCMV3xnlsI-SceI vector and during the 24 h recovery period before re-plating with selection with G418 or G418 + Hyg. We found no differences in either gene conversion or SSA levels in the presence of any of the inhibitors (Figure 4). No differences in gene conversion or SSA levels were found in the UV4DR7 cell line (data not shown). Others and we have previously reported that HR is indeed inhibited by caffeine, KU55933 and CEP-3891 (25,32,33), using other recombination reporter substrates. The difference in these reporter substrates is that they require strand invasion to recover a neo^R gene, and thus SSA does not produce a neo^R gene. Overall, our data does not support a role for checkpoint signalling in SSA.