Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Jul 23;105(30):10320–10325. doi: 10.1073/pnas.0803181105

High mobility group protein B1 enhances DNA repair and chromatin modification after DNA damage

Sabine S Lange *, David L Mitchell *, Karen M Vasquez *,†
PMCID: PMC2492475  PMID: 18650382

Abstract

High mobility group protein B1 (HMGB1) is a multifunctional protein with roles in chromatin structure, transcriptional regulation, V(D)J recombination, and inflammation. HMGB1 also binds to and bends damaged DNA, but the biological consequence of this interaction is not clearly understood. We have shown previously that HMGB1 binds cooperatively with nucleotide excision repair damage recognition proteins to triplex-directed psoralen DNA interstrand cross-links (ICLs). Thus, we hypothesized that HMGB1 modulates the repair of DNA damage in mammalian cells. We demonstrate here that mammalian cells lacking HMGB1 are hypersensitive to DNA damage induced by psoralen plus UVA irradiation (PUVA) or UVC radiation, showing less survival and increased mutagenesis. In addition, nucleotide excision repair efficiency is significantly decreased in the absence of HMGB1 as assessed by the repair and removal of UVC lesions from genomic DNA. We also explored the role of HMGB1 in chromatin remodeling upon DNA damage. Immunoblotting demonstrated that, in contrast to HMGB1 proficient cells, cells lacking HMGB1 showed no histone acetylation upon DNA damage. Additionally, purified HMGB1 protein enhanced chromatin formation in an in vitro chromatin assembly system. These results reveal a role for HMGB1 in the error-free repair of DNA lesions. Its absence leads to increased mutagenesis, decreased cell survival, and altered chromatin reorganization after DNA damage. Because strategies targeting HMGB1 are currently in development for treatment of sepsis and rheumatoid arthritis, our findings draw attention to potential adverse side effects of anti-HMGB1 therapy in patients with inflammatory diseases.

Keywords: HMGB1, nucleotide excision repair, ultraviolet radiation, psoralen, DNA interstand crosslinks

HMGB1 is a highly abundant, multifunctional protein that influences chromatin structure and remodeling by binding to the internucleosomal linker regions in chromatin (1), and facilitating nucleosome sliding (2). In addition, HMGB1 has been shown to be involved in V(D)J recombination (3), transcriptional regulation (4), and in inflammation (5). Because of its role in inflammation, HMGB1 is currently being targeted for treatment of rheumatoid arthritis (6) and sepsis (7), and is also being considered as a player in the invasive and metastatic properties of cancer (8).

Among its diverse functions, HMGB1 is also capable of binding to DNA damaged by carcinogens [e.g., acetylaminofluorene (9), benzo[a]pyrene diol epoxide (9) and UV light (UVC) (10)], and chemotherapeutic agents [e.g., cisplatin (11) and psoralen plus UVA (PUVA) (12)]. Binding of HMGB1 to the damaged site induces a near ninety degree bend in the DNA (11), however, the biological consequence of this interaction is not clear. If by binding to DNA lesions HMGB1 could facilitate DNA repair, the response to DNA damage, or damage-induced chromatin remodeling, then it might prevent a mutagenic and/or carcinogenic outcome of exposure to DNA damaging agents.

The roles of HMGB1 in the response to two distinct types of DNA damage [psoralen DNA interstrand cross-links (ICLs), and 6-4 photoproducts (6-4 PPs) and cyclobutane pyrimidine dimers (CPDs)] are investigated here. Psoralen is a chemotherapeutic agent that, upon UVA irradiation, forms a covalent ICL between both strands of the duplex DNA. This is a complex lesion, and although the mechanism for processing of ICLs in mammalian cells is not yet clearly defined, it is known to involve proteins from at least three major DNA repair pathways: nucleotide excision repair (NER) (13); mismatch repair (14); and homologous recombination (15). The repair of ICLs in cancer cells is a significant determinant of resistance to chemotherapy. HMGB1 is also capable of binding to DNA damage caused by UVC radiation (10), which is a high-energy form of nonionizing radiation that is a known cause of skin cancer. The predominant types of DNA lesions formed by UVC light, 6-4 PPs and CPDs, are repaired exclusively by NER (16). Thus, these lesions represent model substrates for studying the effects of HMGB1 on NER.

Repair of DNA damage becomes more complicated in the context of chromatin, and involves at least three steps (17): (i) initial access to the DNA damage, which is being hindered by the close proximity of nucleosomal proteins; (ii) repair of the damage by the appropriate repair mechanism; and (iii) restoration of the original, predamage chromatin structure. The steps in this “access–repair–restore” model (18) have been well characterized for UV irradiation-induced DNA damage. Access to the UV-lesions is facilitated by immediate acetylation and phosphorylation of core histones H3 and H4 (19–21), and subsequent involvement of chromatin remodeling factors to decondense the DNA-nucleosomal structure (22). During this process, the NER machinery is recruited to the site of damage to remove the lesion. After lesion removal, the original chromatin structure is restored (23). Each step is critical to the maintenance of cellular genetic and epigenetic stability. Given the role of HMGB1 as a known chromatin protein, as well as its ability to bind to damaged DNA, it could play a part in any (or all) of these steps.

We have previously shown that HMGB1 binds to psoralen ICLs cooperatively with XPA-RPA (12), components of the DNA damage/distortion recognition protein complex in the NER mechanism. Because of these interactions, we hypothesized that HMGB1 modulates NER processing of DNA lesions in the cell. In this study, we show that HMGB1 is involved in promoting error-free repair of DNA lesions in mammalian cells. Importantly, the lack of HMGB1 leads to an increase in DNA damage-induced mutagenesis, and reduced cell survival after exposure to DNA damaging agents. We also demonstrate that HMGB1 affects DNA damage-induced chromatin remodeling.

Results

Decrease in Cell Survival in HMGB1 Knockout (KO) MEFs After DNA Damage.

To determine the effect of HMGB1 on cell survival after DNA damage, we measured clonogenic survival of HMGB1 proficient (WT) and deficient (HMGB1 KO) mouse embryonic fibroblasts (MEFs). We found that MEFs lacking HMGB1 had a survival disadvantage after PUVA treatment compared with the WT cells (Fig. 1A), such that at a psoralen concentration of 7.5 × 10−9 M plus 1.8 J/cm2 UVA irradiation, an ≈7-fold difference in clonogenic survival was seen between HMGB1 proficient and deficient cell lines. The KO cells also showed hypersensitivity to UVC irradiation compared with the WT cells (Fig. 1B), such that at a dose of 12 J/m2 UVC irradiation there was an ≈14-fold decrease in clonogenic survival in the absence of HMGB1. Similar effects were also seen in a different matched pair of HMGB1 WT and KO MEFs (data not shown). These results demonstrated that HMGB1 provided a clonogenic survival advantage after exposure to two different types of DNA damaging agents.

Fig. 1.

Fig. 1.

Open in a new tab

Clonogenic survival of WT (black squares) or Hmgb1 KO (gray squares) MEFs after treatment with DNA damaging agents. (A) Survival after treatment with psoralen plus 1.8 J/cm2 UVA irradiation; (B) Survival after treatment with UVC irradiation. Data are means ± SD.

Increase in DNA Damage-Induced Mutagenesis in HMGB1 KO MEFs.

Because the lack of HMGB1 rendered cells more sensitive to DNA damage, we next wanted to determine the extent to which HMGB1 affected the processing of the DNA lesions as assessed by mutagenesis. To determine the effect of HMGB1 on spontaneous or DNA damage-induced mutagenesis, we used a replication-proficient supF mutation-reporter system in KO and WT MEFs. When the supF reporter was damaged by site-specific psoralen ICLs, a 10-fold increase in mutation frequency over background in the WT cell lines occurred (Fig. 2A), as expected (24). Interestingly, the HMGB1 KO MEFs showed a 22-fold increase in psoralen-induced mutations compared with the control (Fig. 2A), demonstrating a 2-fold increase in mutations after ICL damage in MEFs lacking HMGB1 (P < 0.01). UVC irradiation-induced mutagenesis was also significantly higher in the KO MEFs than in WT MEFs (P < 0.01) (Fig. 2B), suggesting that HMGB1 plays a role in suppressing DNA damage-induced mutations. Among the undamaged controls, no difference in spontaneous mutation frequency was detected between the WT and KO cell lines (Fig. 2 A and B).

Fig. 2.

Fig. 2.

Open in a new tab

Mutagenesis in MEFs containing or lacking HMGB1. Spontaneous and DNA damage-induced mutation frequencies in WT or KO MEFs. (A) Plasmids treated with psoralen-modified TFOs and UVA irradiation at 1.8 J/cm2. (B) Plasmids treated with 750 J/m2 UVC irradiation. (C) Mutagenesis in cell-free extracts derived from MEFs that are WT, KO, or supplemented (+HMGB1) with purified recombinant HMGB1. The pSupFG1 plasmid was treated with 500 J/m2 UVC irradiation. *, P < 0.0001; **, P < 0.001; ***, P < 0.05; data are means ± SD.

HMGB1 plays a role in many processes, including transcription. By measuring mutagenesis directly in a cell-free system (i.e., KO and WT cell extracts) supplemented with purified HMGB1 to complement the KO phenotype, we could determine whether the effect of HMGB1 on mutagenic repair was caused directly by its binding to the lesion or by a more indirect effect via its role in transcription regulation. We observed a significant increase in UVC-induced mutagenesis over background in the KO cell extracts compared with that seen both in WT and in HMGB1-supplemented KO extracts (P < 0.05) (Fig. 2C), suggesting that the involvement of HMGB1 in UVC-induced mutagenesis is mediated in a fashion independent of its role in transcription.

To compare the spectrum of mutations induced by psoralen ICLs or UVC-induced DNA damage in the KO cells to those in WT cells, we sequenced DNA from the mutants generated in the MEFs after DNA damage. Even though HMGB1 affected the mutation frequencies, the presence or absence of HMGB1 made no difference in the types of spontaneous or DNA damage-induced mutations. The spontaneous mutations included large deletions and multiple complex mutations [supporting information (SI) Fig. S1 A and B]. Mutations induced by psoralen ICLs were mostly small deletions (1–20 base pairs in length), with <25% of the total mutants being point mutations, typically at or adjacent to the thymine residue to which the ICL was targeted (27 of 29 in WT cells, and 27 of 28 in KO cells) (Fig. S1 C and D). UVC radiation induced a variety of point mutations across the entire mutation-reporter gene. This result was anticipated because formation of this damage was not directed to a specific site; Fig. S1 E and F. The majority of mutations were located at T-T, T-C or C-C residues (sites of 6-4 PP and CPD adduct formation) as expected from translesion-bypass associated DNA damage tolerance (25). These data suggest that, although HMGB1 has an effect on the amount of error-generating-processing of DNA damage generated in the cell, it has no detectable effect on the types of mutations formed during this processing.

HMGB1 Enhances Removal of UVC-Induced DNA Adducts.

HMGB1 is known to influence chromatin structure such that in its absence, the DNA may become more accessible to damaging agents. To determine whether the effects of HMGB1 on cell survival (Fig. 1) and mutagenesis (Fig. 2) after DNA damage were the consequence of the cell's susceptibility to DNA damage or its ability to repair the damage, we subjected WT and KO MEFs to UVC irradiation and compared the amount of 6-4 PPs and CPDs formed in the genomic DNA by using a radioimmunoassay. The amount of DNA damage induced by UVC irradiation in the WT and KO MEFs was not significantly different (Fig. 3 A and B, 0 h), demonstrating that the absence of HMGB1 does not affect the formation of UVC photoproducts on the DNA. To determine the extent to which HMGB1 affected the repair of DNA damage, we measured the removal of UVC-induced DNA lesions over time. At 12 and 24 h after exposure, the WT cells had repaired significantly more 6-4 PPs than the KO cells (2-fold, P < 0.01, and 3-fold, P < 0.01, respectively) (Fig. 3A). A significant decrease (2-fold, P < 0.01) in the repair of CPDs was detected in KO compared with the WT cells 24 h after treatment (Fig. 3B). This result demonstrated that a loss of HMGB1 significantly affects the cell's ability to remove UVC-induced DNA adducts.

Fig. 3.

Fig. 3.

Open in a new tab

Induction and repair of UV-induced DNA damage in MEFs. Quantification of 6-4 photoproducts per megabase (6-4 PPs/MB) (A) or cyclobutane pyrimidine dimers per megabase (CPDs/MB) (B) in WT (black bars) or KO (gray bars) cells after damage with 12.5 J/m2 UV irradiation. *, P < 0.01; **, P < 0.001; data are means ± SEM.

HMGB1 Has a Role in DNA Damage-Induced Chromatin Remodeling.

In addition to the ability of HMGB1 to bind and bend damaged DNA (26), it can also enhance nucleosome sliding (2), suggesting that chromatin remodeling could play a role in HMGB1's effect on DNA repair. As stated above, DNA repair in the context of chromatin is thought to occur in three steps: access to the damage, repair of the damage, and restoration of the original chromatin structure. Acetylation of histones H3 and H4 is a sensitive marker of chromatin accessibility after DNA damage (19). After UV irradiation of WT cells, histone acetylation increased with time, as expected (Fig. 4 A and B). Strikingly, histone acetylation did not increase after DNA damage in the KO cells, suggesting that HMGB1 has a role in chromatin accessibility induced by DNA damage. Total H3 and H4 levels were similar between WT and KO cells before and after DNA damage (data not shown). We have also observed this change in acetylation in another matched pair of HMGB1 KO and WT MEFs (data not shown).

Fig. 4.

Fig. 4.

Open in a new tab

Chromatin remodeling after UVC-irradiation in WT (black bars) or KO (gray bars) MEFs. (A) Representative western blots of histone H3 and H4 acetylation in WT and KO MEFs after 100 J/m2 UVC-irradiation. C is control, untreated cells. (B) Quantification of increase over undamaged control of acetylated histones compared with loading control; data are means ± SEM.

Next, we assessed DNA repair synthesis by measuring nucleotide incorporation into UVC-damaged plasmids onto which chromatin had been assembled in vitro (SI Materials and Methods). In cell-free extracts there was a statistically significant 2-fold increase (P < 0.01) in repair synthesis in the presence of HMGB1 (Fig. S2), demonstrating that HMGB1 enhances DNA repair on chromatinized substrates.

Finally, we explored the effect of HMGB1 on in vitro chromatin assembly. This corresponds to the “restore” step from the access–repair–restore model, and represents the final step of DNA repair in a chromosomal context. We added BSA or HMGB1 to a purified protein chromatin assembly reaction containing human core histones, h-NAP1 and ACF, then measured chromatin formation by supercoiling induced on a relaxed plasmid. The presence of HMGB1 increased the rate of chromatin assembly (Fig. 5) on both undamaged and UVC-damaged plasmid DNA, supporting a role for HMGB1 in chromatin restoration.

Fig. 5.

Fig. 5.

Open in a new tab

Assembly of chromatin in the presence of purified HMGB1. In vitro chromatin assembly on relaxed, undamaged plasmid (A and B) or plasmid irradiated with 5 kJ/m2 UVC (C and D), using purified histones, ACF and h-NAP1, with additional purified BSA or HMGB1 protein. Numbers represent time in minutes. M, marker; SC, supercoiled plasmid; Rlx, relaxed input plasmid. (B and D) Quantification of increased supercoiling in BSA (gray bars) and HMGB1 (black bars) reactions, as a percentage of total DNA loaded; data are means ± SEM.

Discussion

In this article we have demonstrated a role for HMGB1 in cell survival, mutagenesis, DNA repair, and DNA damage-induced chromatin remodeling. In HMGB1 KO MEFs, we observed a decrease in removal of UVC adducts from the DNA, suggesting a role for HMGB1 in the repair of these lesions. This is supported by the finding that the KO MEFs were more sensitive to two different types of DNA damaging agents, psoralen plus UVA irradiation, and UVC irradiation. We also showed that HMGB1 KO cells had a 2-fold increase in mutagenesis upon DNA damage by these same agents, suggesting that HMGB1 specifically contributed to the error-free repair of DNA lesions. It is known that chromatin structure can affect DNA repair, so we explored markers for chromatin remodeling on UVC-induced DNA damage to determine whether this was a mechanism of the effect of HMGB1 on DNA repair. We found that histone acetylation was not induced in the KO cells after UVC irradiation, in striking contrast to the induction seen in WT cells. In addition, HMGB1 was able to enhance the formation of chromatin, as measured by an in vitro chromatin assembly assay. Overall, these results suggest a role for HMGB1 in the repair of UVC and psoralen ICL-induced DNA damage, in a chromosomal context.

A considerable body of work has been published on the in vitro binding of HMGB1 to various damaged and/or distorted DNA substrates (9–12), and all consistently demonstrated a preferential binding of HMGB1 to damaged DNA. Reports of the effect of HMGB1 on the repair of DNA lesions, however, have been much more controversial. Some studies suggest that the binding of HMGB1 to DNA lesions inhibits their repair, in particular of cisplatin-DNA lesions (27–29). This is in contrast to the work presented here, which shows a decrease in DNA repair in the absence of HMGB1. There are several key differences between previous work and ours that could explain this discrepancy. Previous studies were performed on a different type of lesion, cisplatin-DNA, and were executed in vitro in cell-free extracts to which excess amounts of HMGB1 had been added. In contrast, we demonstrated repair of UVC-induced lesions on a chromosomal substrate in cells in the absence of HMGB1. These differences suggest that HMGB1 may act differently at physiological concentrations than when it is over-abundant in an in vitro system. Similar to our work, mismatch repair efficiency has been shown to be decreased in the absence of HMGB1, although this was done in a cell-free system (30).

Recently, an important study published by Prasad et al. (31) demonstrated that HMGB1 could enhance base excision repair (BER) in certain reconstituted systems. They discovered that HMGB1 is capable of binding to 5′dRP sites and that it localizes to sites of base damage with other BER proteins in intact-cell systems. Similar to our work, they demonstrated that HMGB1 could enhance BER, although this was in an in vitro reconstituted system, and was only the case when AP endonuclease was limiting. They showed that HMGB1 KO cells were less sensitive to the DNA damaging agent MMS, but they explained this result by noting that the 5′ dRP BER intermediates were toxic to the cell, and in the absence of HMGB1, AP endonuclease is very slow to remove them. This study is complementary to our work, because it shows a positive effect of HMGB1 on DNA repair by binding to DNA lesions and interacting with repair proteins. Our work adds to this by exploring different types of DNA damage (UVC and psoralen plus UVA) that are repaired by a different repair mechanism (NER, as well as the more complex removal required for the repair of psoralen ICLs). We also provide information about mutagenesis and repair in an intact-cell system, in addition to evidence of a role for HMGB1 in DNA-damage induced chromatin remodeling.

Based on the results presented here, and published work, we suggest a model for the role of HMGB1 in response to DNA damage (Fig. 6). HMGB1 is in equilibrium with histone H1 (32), binding to the entry and exits of nucleosomes. When DNA is damaged, HMGB1 is likely in the vicinity, and binds to and bends the helix at the site of damage. This distortion may assist the NER apparatus in recognizing the damage (12), and facilitating repair of the lesion. HMGB1 also affects chromatin remodeling after DNA damage (Figs. 4 and 5), so its binding to the lesion could increase the accessibility of repair factors to the site of DNA damage.

Fig. 6.

Fig. 6.

Open in a new tab

Model of potential effects of HMGB1 on DNA repair and chromatin remodeling. After DNA damage, HMGB1 binds to the DNA lesion (*), causing a greater distortion in the DNA. It then recruits NER proteins XPA, RPA, and XPC-RAD23B (R23B), as well as chromatin-remodeling factors to remove the nearby nucleosome (N) and facilitate accessibility of the damaged DNA to the repair apparatus.

In conclusion, we have demonstrated that HMGB1 has a role in protecting cells from DNA damage and mutagenesis so that HMGB1 may play a protective role in preventing carcinogenesis. These findings should be considered as a contrast to the suspected role of HMGB1 as an inflammatory promoter of cancer (8), because strategies targeting HMGB1 are in development for treatment of sepsis (7) and rheumatoid arthritis (6) based on its role in inflammation. Our results have implications for any treatment that is designed to target HMGB1, because its depletion may promote carcinogenesis.

Materials and Methods

Cell Lines, Proteins, and Oligonucleotides.

The WT and KO cell lines used in this study were constructed by Calogero et al. (33) with embryonic fibroblasts derived from Hmgb1 WT and KO mice (kindly provided by Marco Bianchi, San Rafaele Research Institute, Milan, Italy). Cells were cultured in 10% FBS-DMEM with penicillin and streptomycin (Invitrogen-Gibco). Whole-cell extracts were made by using the Manley protocol (34). Purified HMGB1 protein was made by using the expression vector pCaln-rHMGB1 (35), kindly provided by Kevin Tracey (North Shore Long Island Jewish Research Institute, Manhasset, New York), designed to express recombinant rat HMGB1 (identical to mouse HMGB1) in E. coli. The 3′-amine-modified triplex-forming oligonucleotides (TFOs) with the psoralen derivative 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) conjugated to the 5′ end were obtained from the Midland Certified Reagent Company. The sequence for the supF-targeting psoralen-conjugated TFO (pAG30) is 5′-pAGGAAGGGGGGGGT GGTGGGGGAGGGGGAG-3′, and the nontargeting scrambled psoralen-conjugated TFO (pSCR30) is 5′-pGGAGGAGTGGAGGGGAGTGAGGGGGGGGGG-3′.

Clonogenic Assay.

Clonogenic assays were carried out as described in Nairn et al. (36). Briefly, cells were treated with a psoralen derivative, HMT (2 × 10−9 M to 1 × 10−8 M; Sigma) for 1 h followed by 1.8 J/cm2 UVA irradiation from a long-wave 365-nm light source after which the media was changed to remove the free psoralen. Alternatively, cells were irradiated with 1 to 30 J/m2 UVC irradiation from a 254-nm light source, then trypsinized, replated, and allowed to grow undisturbed for 8–10 days. Colonies of >100 cells were counted after staining with gentian violet (Fisher). The results were obtained from three independent experiments.

Mutagenesis Assay.

Mutagenesis assays were performed by using the pPySLPT2 supF mutation-reporter shuttle vector, which can replicate in both mouse cells and bacteria due to the presence of polyoma and pBR327 origins of replication, respectively. The assay was performed as described in Christensen et al. (24). Briefly, the pPySLPT2 plasmid was incubated either alone or in the presence of 1 × 10−6 M HMT-conjugated TFO for 16 h at 37°C, followed by treatment with 1.8 J/cm2 long-wave 365-nm UVA irradiation. The TFOs used were either targeted to the supF gene (pAG30) or contained a nontargeting scrambled sequence (pSCR30). Alternatively, we damaged the plasmid with 750 J/m2 UVC, by irradiating in 30 μl of ddH2O under a UVC lamp with a fluence rate of 2.9 J/m2/s. The plasmids were transfected into MEFs with the Amaxa Nucleofector system by using the MEF1 kit and program A23. The cells were left to grow for 48 h, and then the plasmid was isolated by a modified Hirt lysate procedure. After treatment with DpnI to remove unreplicated plasmids, DNA was transformed into MBM7070 [LacZ(Am)] indicator E. coli, and plated on LB-Agar containing 5-bromo-4-chloro-3-indoyl-β-d-galactoside (X-Gal) in the presence of isopropyl-β-d-thiogalactoside (IPTG) and ampicillin. The number of mutant colonies (white) was divided by the total number of colonies (white and blue) to determine a mutation frequency. At least 50,000 colonies were counted for each treatment, and were obtained from three independent experiments. Statistical analyses were performed by using the χ2 test, a sample test of binomial proportion (two-tailed; P < 0.05).

Mutant Characterization.

Mutant colonies were isolated and restreaked onto X-Gal, IPTG, ampicillin plates to confirm a true mutant. DNA was isolated from individual colonies and sequenced by using a primer upstream of the supF promoter.

Cell-Free Extract Mutagenesis Assay.

The mutation-reporter plasmid, pSupFG1, which contains an SV40 origin of replication, was subjected to 500 J/m2 UVC irradiation. 500 ng of damaged or control plasmid was added to 100 μg of cell extract from the MEFs, along with a reaction buffer (30 mM Hepes, pH 7.5, 7 mM MgCl2, 0.5 mM DTT, 4 mM ATP, 100 μM each of dATP, dGTP, dTTP and dCTP, 50 μM each of CTP, GTP, and UTP) and an ATP-regenerating system. Samples of KO extracts were supplemented with 750 ng of purified recombinant rat HMGB1. The reaction was incubated at 37°C for 8 h, and then the DNA was isolated and plated, as in the mutagenesis assay. At least 50,000 colonies were counted for each treatment, and were obtained from three independent experiments. Statistical analyses were done on the fold induction of mutagenesis over no damage control by using a two-sample t test for independent samples with unequal variances (two-tailed; P < 0.05).

Radioimmunoassay.

We carried out this assay as specified in Mitchell et al. (37). Briefly, MEFs were irradiated with 12.5 J/m2 UVC by using a UVC lamp with a fluence rate of 2.9 J/m2/s, and the genomic DNA was isolated at 0, 3, 6, 12, and 24 h. 2 μg of this DNA was placed into duplicate reactions containing radiolabeled, UVC-irradiated dA:dT and a primary antibody that recognizes 6-4 PPs or CPDs (provided by D.L.M.). A secondary antibody was used to pull down the bound DNA, and the total radioactivity was measured by a scintillation counter. We conducted three independent experiments and performed statistical analyses on the number of 6-4 PPs or CPDs/megabase genomic DNA by using the two-sample t test for independent samples with unequal variances (two-tailed; P < 0.05).

Western Blots.

We treated HMGB1 KO and WT MEFS with 100 J/m2 UVC irradiation, then harvested after 1–60 min by scraping on ice in PBS. We pelleted the cells and resuspended in detergent buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.02% NaN3, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM PMSF, protease inhibitors), then sonicated and quantified the protein content of the samples. We subjected 15 μg of protein to electrophoresis on a 15% SDS polyacrylamide gel, and then transferred the proteins to a PVDF membrane, followed by blocking with 5% milk in PBS-T. Primary antibodies were used against histone H3 acetylated at lysines 9 and 14 (1:1500; Upstate), histone H4 acetylated at lysines 5, 8, 12 and 16 (1:1500; Upstate), actin (1:5000; Abcam) and PCNA (1:1500, Santa Cruz Biotechnology). The data were obtained from three independent experiments.

Chromatin Assembly Assay.

To determine the effect of HMGB1 on chromatin assembly on undamaged or UVC-irradiated plasmid DNA, we used the chromatin assembly kit made by Active Motif. We first damaged pTA plasmid with 5 kJ/m2 UVC irradiation, then treated damaged or undamaged plasmid with wheat germ topoisomerase I (Sigma) overnight in a buffer containing 35 mM Tris pH 7.8, 1 mM EDTA, 2 mM DTT, 1.8 mM spermidine, 6.5% glycerol, and 50 μg/ml BSA. We used the chromatin assembly kit to assemble chromatin on the unpurified topoisomerase I-treated DNA according to the manufacturer's instructions, with the addition of either 1 μg of BSA/μg of DNA, or 1 μg of purified recombinant rat HMGB1/μg of DNA. We stopped the reactions at 0.5, 1, 2.5, 5 and 60 min by adding EDTA to a final concentration of 30 μM, then purified the DNA and subjected it to electrophoresis on a 1% agarose, 2 μM chloroquine gel in the absence of EtBr to separate the topoisomers. The gel was stained with Sybr Green I (Molecular Probes) and scanned on a Typhoon variable mode imager (Amersham). Three independent experiments were conducted, and the data were analyzed by comparing the number and intensity of supercoiled bands, after subtraction of the number and intensity of bands in the input plasmid, as a percentage of the total amount of DNA loaded.

Supplementary Material

Supporting Information
supp_105_30_10320__index.html (626B, html)

Acknowledgments.

We thank Ms. Megan Lowery, Mr. Frank Culajay, and Ms. Sarah Henninger for technical assistance and Drs. Rick Finch and Richard Wood for useful discussions. This work was supported by National Institutes of Health/National Cancer Institute Grants CA097175 and CA093729 (to K.M.V.), a National Institute of Environmental Health Sciences Grant ES07784, and an American Legion Auxiliary fellowship (to S.S.L.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0803181105/DCSupplemental.

References

  • 1.Nightingale K, Dimitrov S, Reeves R, Wolffe AP. Evidence for a shared structural role for HMG1 and linker histones B4 and H1 in organizing chromatin. EMBO J. 1996;15:548–561. [PMC free article] [PubMed] [Google Scholar]
  • 2.Bonaldi T, et al. The DNA chaperone HMGB1 facilitates ACF/CHRAC-dependent nucleosome sliding. EMBO J. 2002;21:6865–6873. doi: 10.1093/emboj/cdf692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Swanson PC. Fine structure and activity of discrete RAG-HMG complexes on V(D)J recombination signals. Mol Cell Biol. 2002;22:1340–1351. doi: 10.1128/mcb.22.5.1340-1351.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Boonyaratanakornkit V, et al. High-mobility group chromatin proteins 1 and 2 functionally interact with steroid hormone receptors to enhance their DNA binding in vitro and transcriptional activity in mammalian cells. Mol Cell Biol. 1998;18:4471–4487. doi: 10.1128/mcb.18.8.4471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Andersson U, et al. High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med. 2000;192:565–570. doi: 10.1084/jem.192.4.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ulloa L, et al. High mobility group box chromosomal protein 1 as a nuclear protein, cytokine, and potential therapeutic target in arthritis. Arthritis Rheum. 2003;48:876–881. doi: 10.1002/art.10854. [DOI] [PubMed] [Google Scholar]
  • 7.Wang H, et al. HMGB1 as a late mediator of lethal systemic inflammation. Am J Respir Crit Care Med. 2001;164:1768–1773. doi: 10.1164/ajrccm.164.10.2106117. [DOI] [PubMed] [Google Scholar]
  • 8.Ellerman JE, et al. Masquerader: High mobility group box-1 and cancer. Clin Cancer Res. 2007;13:2836–2848. doi: 10.1158/1078-0432.CCR-06-1953. [DOI] [PubMed] [Google Scholar]
  • 9.Lanuszewska J, Widlak P. High mobility group 1 and 2 proteins bind preferentially to DNA that contains bulky adducts induced by benzopyrene diol epoxide and N-acetoxy-acetylaminofluorene. Cancer Lett. 2000;158:17–25. doi: 10.1016/s0304-3835(00)00517-6. [DOI] [PubMed] [Google Scholar]
  • 10.Pasheva EA, Pashev IG, Favre A. Preferential binding of high mobility group 1 protein to UV-damaged DNA. Role of the COOH-terminal domain. J Biol Chem. 1998;273:24730–24736. doi: 10.1074/jbc.273.38.24730. [DOI] [PubMed] [Google Scholar]
  • 11.Ohndorf UM, et al. Basis for recognition of cisplatin-modified DNA by high-mobility-group proteins. Nature. 1999;399:708–712. doi: 10.1038/21460. [DOI] [PubMed] [Google Scholar]
  • 12.Reddy MC, Christensen J, Vasquez KM. Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA. Biochemistry. 2005;44:4188–4195. doi: 10.1021/bi047902n. [DOI] [PubMed] [Google Scholar]
  • 13.Vasquez KM, et al. Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions. Proc Natl Acad Sci USA. 2002;99:5848–5853. doi: 10.1073/pnas.082193799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Wu Q, Christensen LA, Legerski RJ, Vasquez KM. Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells. EMBO Rep. 2005;6:551–557. doi: 10.1038/sj.embor.7400418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Cipak L, Watanabe N, Bessho T. The role of BRCA2 in replication-coupled DNA interstrand cross-link repair in vitro. Nat Struct Mol Biol. 2006;13:729–733. doi: 10.1038/nsmb1120. [DOI] [PubMed] [Google Scholar]
  • 16.Sinha RP, Hader DP. UV-induced DNA damage and repair: A review. Photochem Photobiol Sci. 2002;1:225–236. doi: 10.1039/b201230h. [DOI] [PubMed] [Google Scholar]
  • 17.Green CM, Almouzni G. When repair meets chromatin. First in series on chromatin dynamics. EMBO Rep. 2002;3:28–33. doi: 10.1093/embo-reports/kvf005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Smerdon MJ. DNA repair and the role of chromatin structure. Curr Opin Cell Biol. 1991;3:422–428. doi: 10.1016/0955-0674(91)90069-b. [DOI] [PubMed] [Google Scholar]
  • 19.Ramanathan B, Smerdon MJ. Changes in nuclear protein acetylation in u. v.-damaged human cells. Carcinogenesis. 1986;7:1087–1094. doi: 10.1093/carcin/7.7.1087. [DOI] [PubMed] [Google Scholar]
  • 20.Zhong SP, Ma WY, Dong Z. ERKs and p38 kinases mediate ultraviolet B-induced phosphorylation of histone H3 at serine 10. J Biol Chem. 2000;275:20980–20984. doi: 10.1074/jbc.M909934199. [DOI] [PubMed] [Google Scholar]
  • 21.Zhong S, et al. Ultraviolet B-induced phosphorylation of histone H3 at serine 28 is mediated by MSK1. J Biol Chem. 2001;276:33213–33219. doi: 10.1074/jbc.M103973200. [DOI] [PubMed] [Google Scholar]
  • 22.Mitchell DL, Clarkson JM, Adair GM. The DNA of UV-irradiated normal and excision-deficient mammalian cells undergoes relaxation in an initial stage of DNA repair. Mutat Res. 1986;165:123–128. doi: 10.1016/0167-8817(86)90068-4. [DOI] [PubMed] [Google Scholar]
  • 23.Gaillard PH, et al. Chromatin assembly coupled to DNA repair: A new role for chromatin assembly factor I. Cell. 1996;86:887–896. doi: 10.1016/s0092-8674(00)80164-6. [DOI] [PubMed] [Google Scholar]
  • 24.Christensen LA, Conti CJ, Fischer SM, Vasquez KM. Mutation frequencies in murine keratinocytes as a function of carcinogenic status. Mol Carcinog. 2004;40:122–133. doi: 10.1002/mc.20026. [DOI] [PubMed] [Google Scholar]
  • 25.Todd PA, Glickman BW. Mutational specificity of UV light in Escherichia coli: Indications for a role of DNA secondary structure. Proc Natl Acad Sci USA. 1982;79:4123–4127. doi: 10.1073/pnas.79.13.4123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Bustin M, Reeves R. High-mobility-group chromosomal proteins: Architectural components that facilitate chromatin function. Prog Nucleic Acid Res Mol Biol. 1996;54:35–100. doi: 10.1016/s0079-6603(08)60360-8. [DOI] [PubMed] [Google Scholar]
  • 27.Huang JC, et al. HMG-domain proteins specifically inhibit the repair of the major DNA adduct of the anticancer drug cisplatin by human excision nuclease. Proc Natl Acad Sci USA. 1994;91:10394–10398. doi: 10.1073/pnas.91.22.10394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Zamble DB, et al. Repair of cisplatin–DNA adducts by the mammalian excision nuclease. Biochemistry. 1996;35:10004–10013. doi: 10.1021/bi960453+. [DOI] [PubMed] [Google Scholar]
  • 29.Malina J, Kasparkova J, Natile G, Brabec V. Recognition of major DNA adducts of enantiomeric cisplatin analogs by HMG box proteins and nucleotide excision repair of these adducts. Chem Biol. 2002;9:629–638. doi: 10.1016/s1074-5521(02)00134-5. [DOI] [PubMed] [Google Scholar]
  • 30.Yuan F, et al. Evidence for involvement of HMGB1 protein in human DNA mismatch repair. J Biol Chem. 2004;279:20935–20940. doi: 10.1074/jbc.M401931200. [DOI] [PubMed] [Google Scholar]
  • 31.Prasad R, et al. HMGB1 is a cofactor in mammalian base excision repair. Mol Cell. 2007;27:829–841. doi: 10.1016/j.molcel.2007.06.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Catez F, et al. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24:4321–4328. doi: 10.1128/MCB.24.10.4321-4328.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Calogero S, et al. The lack of chromosomal protein Hmg1 does not disrupt cell growth but causes lethal hypoglycaemia in newborn mice. Nat Genet. 1999;22:276–280. doi: 10.1038/10338. [DOI] [PubMed] [Google Scholar]
  • 34.Manley JL, Fire A, Samuels M, Sharp PA. In vitro transcription: Whole-cell extract. Methods Enzymol. 1983;101:568–582. doi: 10.1016/0076-6879(83)01038-1. [DOI] [PubMed] [Google Scholar]
  • 35.Wang H, et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science. 1999;285:248–251. doi: 10.1126/science.285.5425.248. [DOI] [PubMed] [Google Scholar]
  • 36.Nairn RS, et al. UV mutagenesis, cytotoxicity and split-dose recovery in a human-CHO cell hybrid having intermediate (6-4) photoproduct repair. Mutat Res. 1989;217:193–201. doi: 10.1016/0921-8777(89)90071-2. [DOI] [PubMed] [Google Scholar]
  • 37.Mitchell DL. Quantification of DNA photoproducts in mammalian cell DNA using radioimmunoassay. In: Daryl S. Henderson., editor. Methods in Molecular Biology, DNA Repair Protocols. 2nd Edition. Totowa, NJ: The Humana Press, Inc.; 2006. pp. 239–249. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
supp_105_30_10320__index.html (626B, html)
0803181105_0803181105SI.pdf (526.1KB, pdf)

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES