Homologous recombination but not nucleotide excision repair plays a pivotal role in tolerance of DNA-protein cross-links in mammalian cells - PubMed (original) (raw)

. 2009 Oct 2;284(40):27065-76.

doi: 10.1074/jbc.M109.019174. Epub 2009 Aug 11.

Atsushi Katafuchi, Mayumi Matsubara, Hiroaki Terato, Tomohiro Tsuboi, Tasuku Masuda, Takahiro Tatsumoto, Seung Pil Pack, Keisuke Makino, Deborah L Croteau, Bennett Van Houten, Kenta Iijima, Hiroshi Tauchi, Hiroshi Ide

Affiliations

Toshiaki Nakano et al. J Biol Chem. 2009.

Abstract

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.

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Figures

FIGURE 1.

FIGURE 1.

Upper size limit of CLPs amenable to mammalian NER is 8–10 kDa in vitro. A, partial sequence of substrates (150OXA-DPCs). Proteins listed in

supplemental Table S1

were tethered to oxanine (O). The arrows indicate the incision sites with HeLa CFEs. B, PAGE analysis of incision products. Left, analysis of 5′ incision. 150OXA-DPCs (5′-end 32P-labeled) were incubated with HeLa CFEs (100 μg) for 30 min. After incubation, samples were treated with proteinase K and separated by 10% denaturing PAGE. Right, analysis of 3′ incisions. 150OXA-DPCs (3′-end 32P-dC-labeled) were treated, and products were analyzed as on the left. The leftmost lanes (M) indicate 59-mer and 64-mer markers in the left and right panels, respectively. C, variations of the DPC incision efficiency with the size of CLPs. Left, incision with HeLa CFEs. The amounts of 5′-nicked products were quantified from the left panel in B and

supplemental Fig. S2

and are plotted against the size of CLPs. Right, incision with UvrABC. 60OXA-DPCs were incubated with B. caldotenax UvrA and UvrB and T. maritima UvrC for 30 min at 37 or 55 °C. Products were analyzed by 12% denaturing PAGE (not shown). The amounts of 5′-nicked products are plotted against the size of CLPs.

FIGURE 2.

FIGURE 2.

Chromosomal DPCs are not removed by NER in mammalian cells. A, scheme for analysis of FA-induced CLPs and CLXs using SDS-PAGE (a) and fluorescence labeling with FITC (b) and MDPF (c). B, SDS-PAGE analysis of CLPs. WT (WI38VA13) and XPA (XP12ROSV) cells were treated with 0.5 m

m

FA for 3 h, and chromosomal DNA was isolated after the indicated period of repair incubation. CLPs in 40 μg of DNA were released by heat and separated by 10% SDS-PAGE. Bands were visualized by silver staining. The two leftmost lanes show size markers. C, release of CLPs measured with FITC. WT, XPA, and XPC (XP4PASV) cells were treated with FA, and DNA was isolated as in B. CLPs in 15 μg of DNA were labeled by FITC, and their fluorescence was measured. Data points are means of two independent experiments. D, release of small adducts (CLXs) measured with MDPF. WT and XPA cells were treated with FA, and DNA was isolated as in B. Cross-linked adducts in 150 μg of DNA were released by heat and fractionated by dialysis (3.5 kDa cut-off). The adducts (>3.5 and <3.5 kDa) were separately labeled by MDPF, and their fluorescence was measured. Note that the fraction with >3.5 kDa contained CLPs and the large component of CLXs, whereas that with <3.5 kDa contained the small component of CLXs. Data points are means of two independent experiments. E, survival of WT and XPA (XP12ROSV and XP2OSSV) cells treated with FA (left) or azadC (right). Cells were incubated with the indicated concentrations of FA for 3 h or azadC for 24 h. Cell survival was assayed by colony formation. Data points are means of three independent experiments.

FIGURE 3.

FIGURE 3.

CLPs are not polyubiquitinated for proteasomal degradation. A, protocols of cell treatment. B, Western blotting analysis of polyubiquitinated CLPs. In the presence or absence of 10 μ

m

MG132, WI38VA13 cells were treated with 0.5 m

m

FA for 3 h and subjected to repair incubation for 4 h as shown in A. DNA (50 μg) isolated from mock-treated cells (−FA; lanes 1, 5, and 6) or FA-treated cells (+FA; lanes 7 and 8) were slot-blotted on a membrane and probed with polyubiquitin-specific FK1 antibodies. Lysozyme (Lyso) and ubiquitinated lysozyme ((Ub)n-Lyso)) were also slot-blotted as control and analyzed similarly (lanes 2–4). C, SDS-PAGE analysis of polyubiquitinated CLPs. WI38VA13 cells were treated with 0.5 m

m

FA in the presence of 10 μ

m

MG132 as described in B. DNA (50 μg) isolated from cells was heated to release CLPs and mixed with an affinity matrix for ubiquitinated proteins. The supernatant was removed (unbound fraction), and the matrix was washed twice (wash-1 and wash-2 fractions). Finally, bound proteins were eluted with SDS-loading buffer (elute fraction). Proteins were separated by 10% SDS-PAGE and visualized by silver staining. The two leftmost lanes (M) show size markers.

FIGURE 4.

FIGURE 4.

DPCs induce nuclear RAD51 and γ-H2AX foci. MRC5SV cells were treated with 0.1 m

m

FA for 3 h or 1 μ

m

azadC for 24 h and subjected to repair incubation for the indicated periods. Cells were fixed, probed with RAD51 and γ-H2AX antibodies, and analyzed for nuclear foci. A, FA treatment. B, azadC treatment. The upper panels show the fraction of foci-positive cells, and lower panels show the number of foci/cell. Cells containing more than 10 RAD51 or 20 γ-H2AX foci were counted as foci-positive cells. Data points are means of three or four independent experiments with S.D.

FIGURE 5.

FIGURE 5.

DPCs result in accumulation of DSBs in HR-deficient but not HR-proficient cells. AA8 (WT) and irs1SF (XRCC3) cells were treated with azadC (1 μ

m

, 24 h) or FA (0.2 m

m

, 3 h) and incubated without azadC for 12 h or FA for 6 h. Tail moment as a measure of strand breaks was analyzed by neutral and alkaline comet assays as described under “Experimental Procedures.” Data points are means of three independent experiments with S.D. A, azadC treatment. B, FA treatment. The upper and lower panels show the results of neutral and alkaline comet assays, respectively.

FIGURE 6.

FIGURE 6.

DPCs induce phosphorylation of CHK1 and CHK2. A, protocols of cell treatment with UV, FA, and azadC. Broken lines indicate post-repair incubation without damaging agents, and WB indicates the time point where the sample was taken for Western blotting analysis. B, Western blotting analysis of phosphorylation of CHK1 and CHK2. WI38VA13 cells were treated with FA (0.4 m

m

for 3 h), azadC (2 μ

m

for 24 h), or UV (50 J/m2). After the indicated periods of treatment or repair incubation, cells were collected and lysed. Proteins (150 μg) were separated by 10% SDS-PAGE; blotted on a membrane; and probed with CHK1, CHK2, phospho-CHK1 (Ser317), phospho-CHK2 (Ser19), and actin antibodies.

FIGURE 7.

FIGURE 7.

Responses of mammalian cells to DPCs. DPCs induce fork stalling and rapidly activate the ATR damage response pathway. Subsequently, forks stalled at DPCs undergo breakage, initiating HR that reestablishes replication forks. It is not clear whether the ATM damage response pathway is activated by fork breakage (DSBs) or ATR. Neither NER nor NER coupled with the proteasomal degradation of CLPs participates in the repair of DPCs. DSBs resulting from fork breakage are not repaired by NHEJ.

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