Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase - PubMed (original) (raw)

Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase

Yuichiro Shimizu et al. EMBO J. 2003.

Abstract

The XPC-HR23B complex recognizes various helix-distorting lesions in DNA and initiates global genome nucleotide excision repair. Here we describe a novel functional interaction between XPC-HR23B and thymine DNA glycosylase (TDG), which initiates base excision repair (BER) of G/T mismatches generated by spontaneous deamination of 5-methylcytosine. XPC-HR23B stimulated TDG activity by promoting the release of TDG from abasic sites that result from the excision of mismatched T bases. In the presence of AP endonuclease (APE), XPC-HR23B had an additive effect on the enzymatic turnover of TDG without significantly inhibiting the subsequent action of APE. Our observations suggest that XPC-HR23B may participate in BER of G/T mismatches, thereby contributing to the suppression of spontaneous mutations that may be one of the contributory factors for the promotion of carcinogenesis in xeroderma pigmentosum genetic complementation group C patients.

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Figures

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Fig. 1. Physical interaction of XPC–HR23B and TDG. Glutathione–Sepharose beads (20 µl) were incubated in 100 µl of the binding mixture with 16.6 nM of either GST (lane 3) or GST–TDG (lanes 4–6) in the presence of XPC–HR23B (6.7 nM). For lanes 5 and 6, 166 nM and 1.66 µM His-TDG, respectively, were also included as a competitor. After extensive washing, bound proteins were eluted with buffer containing 10 mM glutathione. A quarter of each eluate was mixed with whole-cell extract from XP4PASV cells, which do not express XPC, and subjected to 8% SDS–PAGE followed by immunoblotting with anti-XPC antibody. Lane 1, 0.4% of the input XPC; lane 2, XP4PASV cell extract only.

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Fig. 2. DNA substrates used in gel mobility shift analysis (A) and the nicking assays (B). X is A, C, G or T.

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Fig. 3. XPC–HR23B is recruited to TDG-bound DNA and forms a ternary complex. (A) The indicated concentrations of XPC–HR23B or TDG were incubated at 30°C for 30 min with 0.35 nM of the 32P-labeled DNA fragment containing a single G/T mismatch or completely paired double-stranded DNA (G/C) as a control. The resulting DNA–protein complex was fixed with glutaraldehyde, and resolved by native PAGE. (B) The DNA substrate containing a single G/T mismatch (0.35 nM) was incubated at 30°C for 15 min in the presence or absence of His-TDG (16 nM). The indicated concentrations of XPC–HR23B were then added, incubated further at 30°C for 15 min and subjected to native PAGE after glutaraldehyde fixation. The asterisk shows the bands that newly appeared in the presence of both TDG and XPC–HR23B. (C) A supershift assay identifying the ternary complex containing TDG, XPC–HR23B and DNA. Sequential binding reactions were conducted as in (B) with 16 nM TDG, 6.7 nM XPC–HR23B and 0.35 nM DNA substrate containing a single G/T mismatch. Various amounts of anti-TDG or anti-XPC antibodies were then added and incubated further on ice for 15 min before cross-linking the DNA–protein complexes with glutaraldehyde.

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Fig. 4. XPC–HR23B stimulates the activity of TDG. (A) A nicking assay measuring the TDG activity in the presence of XPC–HR23B. The 60mer DNA substrate containing a single G/T mismatch, which was labeled at the 5′ end of the T-strand, was incubated at 30°C for the indicated time with 0.42 nM His-TDG in the presence or absence of various concentrations of XPC–HR23B. The DNA samples were purified and subjected to alkali treatment to cleave the resulting AP sites, after which denaturing PAGE was performed. (B) The percentage of the cleaved oligonucleotides in the 32P-labeled DNA substrate was calculated for each lane in (A) and plotted as a graph. The mean values and standard errors were calculated from at least two independent experiments. (C) A nicking assay using 0.42 nM His-TDG in the presence of various concentrations of XPC–HR23B (lane 2), XPC alone (lanes 3–6) or HR23B alone (lanes 7–10) as indicated. All reactions were incubated at 30°C for 120 min and the purified DNA samples were subjected to alkali treatment and denaturing PAGE.

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Fig. 5. XPC–HR23B promotes the turnover of TDG. (A) The 32P-labeled DNA substrate containing a single G/T mismatch was incubated with 0.42 nM His-TDG at 30°C for various periods as indicated (lanes 1–5). After 120 min, the indicated concentrations of XPC–HR23B were added and incubated further at 30°C for 15, 30 or 60 min (total incubation time is shown above each lane). The purified DNA samples were subjected to alkali treatment and denaturing PAGE. (B) The percentage of the cleaved oligonucleotides in the 32P-labeled DNA substrate was calculated for each lane in (A). The mean values and standard errors were calculated from at least two independent experiments.

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Fig. 6. XPC–HR23B stimulates the activity of TDG in the presence of APE. (A) The 32P-labeled DNA substrate containing a single G/T mismatch was incubated with 0.42 nM His-TDG, 2.1 nM His-APE and the indicated concentrations of XPC–HR23B at 30°C for the specified time. The purified DNA samples were subjected to alkali treatment and denaturing PAGE. Due to the 3′→5′ exonuclease activity of APE, the bands of cleaved oligonucleotides appear as ladders. (B) The percentage of the cleaved oligonucleotides in the 32P-labeled DNA substrate was calculated for each lane in (A). The mean values and standard errors were calculated from at least two independent experiments.

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Fig. 7. XPC–HR23B does not inhibit the enzymatic action of APE. (A) The 32P-labeled DNA substrate containing a synthetic AP site analog opposite four different bases or a normal G/C pair at the corresponding position (0.35 nM each) were incubated at 30°C for 30 min with the indicated concentrations of XPC–HR23B. The resulting DNA–protein complex was fixed with glutaraldehyde, and resolved by native PAGE. (B) The 32P-labeled DNA substrate containing a single G/T mismatch was incubated with 0.42 nM His-TDG, 2.1 nM His-APE and the indicated concentrations of XPC–HR23B at 30°C for the specified periods. The reactions were stopped by addition of EDTA and subjected to denaturing PAGE without alkali treatment. (C) The percentage of the cleaved oligonucleotides in the 32P-labeled DNA substrate was calculated for each lane in (B). The mean values and standard errors were calculated from at least two independent experiments.

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Fig. 8. A schematic model depicting a possible mechanism for TDG stimulation by XPC–HR23B. A G/T mismatch can be generated by spontaneous deamination of 5-methylcytosine (I). After the removal of the mismatched T (II), TDG remains attached to the resulting AP site through interaction with the opposite guanine residue. XPC–HR23B is then recruited via protein–protein interaction and/or structural changes of DNA induced by TDG (III). It has been shown that XPC–HR23B preferentially recognizes DNA secondary structure containing a junction with a single-stranded arm, branching away from duplex DNA in the 3′ direction (Sugasawa et al., 2002). Therefore, XPC–HR23B probably interacts with the unpaired guanine and binds to the 3′ side of the AP site. XPC–HR23B may thus compete with TDG for the guanine residue and push it out from the AP site (IV).

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