Human HMGB1 directly facilitates interactions between nucleotide excision repair proteins on triplex-directed psoralen interstrand crosslinks

Abstract

Psoralen is a chemotherapeutic agent that acts by producing DNA interstrand crosslinks (ICLs), which are especially cytotoxic and mutagenic because their complex chemical nature makes them difficult to repair. Proteins from multiple repair pathways, including nucleotide excision repair (NER), are involved in their removal in mammalian cells, but the exact nature of their repair is poorly understood. We have shown previously that HMGB1, a protein involved in chromatin structure, transcriptional regulation, and inflammation, can bind cooperatively to triplex-directed psoralen ICLs with RPA, and that mammalian cells lacking HMGB1 are hypersensitive to psoralen ICLs. However, whether this effect is mediated by a role for HMGB1 in DNA damage recognition is still unknown. Given HMGB1's ability to bind to damaged DNA and its interaction with the RPA protein, we hypothesized that HMGB1 works together with the NER damage recognition proteins to aid in the removal of ICLs. We show here that HMGB1 is capable of binding to triplex-directed psoralen ICLs with the dedicated NER damage recognition complex XPC-RAD23B, as well as XPA-RPA, and that they form a higher-order complex on these lesions. In addition, we demonstrate that HMGB1 interacts with XPC-RAD23B and XPA in the absence of DNA. These findings directly demonstrate interactions between HMGB1 and the NER damage recognition proteins, and suggest that HMGB1 may affect ICL repair by enhancing the interactions between NER damage recognition factors.

Figure 1. Interactions of human HMGB1 and XPC-RAD23B proteins on triplex-directed psoralen ICLs

Human purified recombinant HMGB1 and XPC-RAD23B proteins (~1 × 10⁻⁸ M) were incubated with ~1 × 10⁻⁸ M radiolabeled triplex-directed psoralen ICLs in binding buffer for 20 min at 30°C. Antibodies were added to indicated samples and incubation was continued for an additional 20 min. Protein-DNA complexes were subjected to EMSA on a 6% native PAGE gel and visualized by autoradiography. Lane 1 contains non-damaged duplex DNA, and the remaining lanes contain triplex-directed psoralen ICLs (Tdp-ICL substrate).

Figure 2. Binding of human purified recombinant HMGB1, XPC-RAD23B, and RPA proteins on triplex-directed psoralen ICLs

Human purified recombinant HMGB1, XPC-RAD23B, and RPA proteins (~1 × 10⁻⁸ M) were incubated with ~1 × 10⁻⁸ M radiolabeled triplex-directed psoralen ICLs in binding buffer for 20 min at 30°C. Antibodies were added to the indicated samples and incubation was continued for an additional 20 minutes. Protein-DNA complexes were subjected to EMSA on a 6% native PAGE gel and visualized by autoradiography. Lane 1 contains non-damaged duplex DNA, and the remaining lanes contain triplex-directed psoralen ICLs (Tdp-ICL substrate).

Figure 3. Identification of proteins in the protein-DNA complexes

(A) Human purified recombinant HMGB1, XPC-RAD23B, and RPA proteins (~1 × 10⁻⁸ M) were incubated with ~1 × 10⁻⁸ M radiolabeled triplex-directed psoralen ICLs (lanes 1, 3 and 5), or in the absence of DNA (lanes 2, 4 and 6) in binding buffer for 20 min at 30°C. Protein-DNA complexes were subjected to EMSA on a 6% native PAGE gel and visualized by autoradiography, followed by semi-dry blotting to transfer the protein-DNA complexes to a membrane. The membrane was then blotted with an α-RPA-70 antibody; (B, C) Human purified recombinant HMGB1 (lane 1), XPC-RAD23B (lane 2) and RPA (lane 3) were incubated separately, or together (lane 4), on a triplex-directed psoralen ICL substrate for 20 min at 30°C, followed by EMSA analysis using a 6% native PAGE gel (B). The electrophoretic bands corresponding to the protein-DNA complexes were excised, subjected to SDS-PAGE, and immunoblotted with antibodies against XPC, RPA and HMGB1 (C).

Figure 4. Sequential addition of human purified recombinant HMGB1, XPC-RAD23B, and RPA to triplex-directed psoralen ICLs

(A) Purified recombinant human HMGB1 (10 nM) was incubated for 20 minutes at 30°C with radiolabeled triplex-directed psoralen ICLs, after which increasing concentrations of XPC-RAD23B and RPA proteins (10, 20, or 30 nM) were added, incubated for a further 20 minutes, and then subjected to EMSA analysis using a 6% native PAGE. (B) Recombinant human XPC-RAD23B and RPA proteins (10 nM) were incubated for 20 minutes with the radiolabeled ICL-containing substrate (Tdp-ICL substrate), followed by incubation with increasing concentrations of HMGB1 (10, 20, or 30 nM) for 20 minutes, then analyzed using EMSA.

Figure 5. Interactions between HMGB1 and NER DNA damage recognition factors

(A, B) Calmodulin resin was bound with CBP-tagged HMGB1 protein, then incubated with cell extracts (A) or human purified XPC, HMGB1, and RPA proteins (B), and the bound proteins were electrophoresed by SDS-PAGE, transferred to membranes and immunoblotted with antibodies against HMGB1 or XPC. Calmodulin-resin (CaM resin) alone was used as a specificity control, and cell extracts (Cell Ext, A) or purified proteins (Prot Mark, B) were run on the gels as protein-size markers. (C, D) Amylose resin was bound with MBP-tagged XPC-RAD23B protein (C), or XPA protein (D) then incubated with cell extracts (C) or human purified HMGB1 and RPA proteins (D), and then immunoblotted as in A. MBP peptide (C) or amylose-resin (Amyl resin, D) alone were used as specificity controls, and cell extracts (Cell Ext, C) or purified proteins (Prot Mark, D) were run on the gels as protein-size markers. (E) Representative immunoblots of human XPA and HMGB1 proteins immunopreciptated from cell extracts using antibodies against rabbit IgG (control) and HMGB1.