HMGB1: roles in base excision repair and related function

Abstract

High mobility group box 1 (HMGB1) is a nonhistone architectural protein that is involved in many biological processes including chromatin remodeling, transcription, cell signaling of inflammation, DNA damage repair and others. Recent studies have identified the cross-link of HMGB1 with a DNA base excision repair intermediate indicating that this protein is involved in base excision repair (BER) pathway. Further characterization of the roles of HMGB1 in BER demonstrates that the protein acts as a cofactor to regulate BER sub-pathways by inhibiting single-nucleotide BER and stimulating long-patch BER through modulating the activities of base excision repair enzymes. Directing of base lesion repair to the long-patch sub-pathway can result in trinucleotide repeat instability suggesting an important role of HMGB1 in modulating genome stability.

Figure 1

Structures of HMGB1 protein domains. Tertiary structures of HMGB1 Box A-DNA and Box B were produced according to published data (refs. and 9) using Chimera [58].

Figure 2

Photoaffinity labeling of HMGB1 in Cell extracts (from Figure S2 of ref. 23). (A) Diagram of BER intermediate with radiolabeled uracil and the scheme of enzymatic reactions. (B) NaBH₄ crosslinking of proteins in various cell extracts with the substrate treated by UDG.

Figure 3

NaBH₄ Crosslinking of HMGB1 in Cell Extracts (from Figure 1 of ref. 23). NaBH₄ crosslinking of proteins in various cell extracts with the UDG-untreated substrate.

Figure 4

Accumulation of GFP-tagged Proteins at Sites of DNA damage in Living Cells (from Figure 3 of ref. 15). The recruitment of GFP-tagged human OGG1, NITH1, HMGB1, Ku70 and Rad52 on DNA damage induced by 405 nm scanning laser in the absence and presence of photosensitizer (8-MOP).

Figure 5

Characterization of HMGB1 Isolated from HeLa Cells (from Figure 4 of ref. 23). (A) Schematic representation of the dRP lyase substrate generated by treatment of the radiolabeled oligonucleotide substrates with UDG and APE1. (B) The dRP lyase activities of Pol β and HMGB1. (C) The purified HMGB1 (lane 2 and 3, 0.45 and 0.9 μg, respectively) was renatured (lane 4, ~0.2 μg) and analyzed by NuPAGE Bis-Tris gel electrophoresis. (D) NaBH₄ crosslinking of renatured HMGB1 at the concentrations of 500 nM (lane 2), 140 nM (lane 3) and 280 nM (lane 4). (E) The dRP lyase activity of renatured HMGB1 at concentration of 420 nM. (F) Quantification of the dRP lyase activities of purified (open circles) and renatured HMGB1 (filled circles). (G) The effects of HMGB1 on single-nucleotide BER.

Figure 6

HMGB1 Binding to BER Intermediates (from Figure S4 of ref. 23) (A) HMGB1 binding to the BER intermediate with a single-nucleotide gap and a sugar phosphate measured by gel mobility shift assay. (B) Quantification of the HMGB1-DNA complex was plotted as a function of HMGB1 concentration. (C) The binding affinity (apparent K_d) of HMGB1 on BER intermediate DNA substrates

Figure 7

Biological and Biochemical Analysis of HMGB1^+/+ and HMGB1^−/− MEFs after Treatment with MMS (from Figure 7 of ref. 23). (A) MMS sensitivity of HMGB1^+/+ (red open circles) and HMGB1^−/− (blue open circles) cells. (B) MMS sensitivity of HMGB1^+/+ (red filled symbols) and HMGB1^−/− (blue open symbols) cells in the absence (circles) and presence of MX for 1 (squares) or 4 hr (diamonds). (C) Comet assay of MMS-induced single-strand DNA breaks in HMGB1^+/+ and HMGB1^−/− cells in the absence and presence of MX. (D) Quantification of Olive tail moment (OTM) of comet from (C) in HMGB1^+/+ (red bars) and HMGB1^−/− cells (blue bar). (E) HMGB1 stimulates APE1 incision on MX-adducted DNA substrate. (F) Quantification of HMGB1 stimulatory effects on APE1 incision activity.

Figure 8

Stimulation of APE1 Activity by HMGB1 (from Figure 5 of ref. 23) (A) HMGB1 stimulates APE1 incision activity on normal AP site. (B) Quantification of the HMGB1 stimulatory effects on APE1.

Figure 9

Stimulation of FEN1 by HMGB1 (from Figure 5 of ref. 23). (A) The effect of HMGB1 on FEN1 cleavage activity on a nicked-3nt flap substrate. (B) Quantification of FEN1 activity in (A). (C) HMGB1 stimulates FEN1 cleavage on a nicked-THF flap substrate. (D) The quantification of FEN1 activity in (C).

Figure 10

HMGB1 Facilitates CAG Repeat Expansion (from Figure 6 of ref. 48). (A) Repair products from BER reactions mediated by HMGB1^+/+ and HMGB1^−/− cell extracts in the context of CAG repeats. (B) Quantification of expansion products from HMGB1^+/+ and HMGB1^−/− cell extracts.

Figure 11

Recombinant HMGB1 Stimulates CAG Repeat Expansion (From Figure 6 of ref. 48). The effect of HMGB1 on CAG repeat expansion was determined by addition of purified HMGB1 to the HMGB1 null cell extract. Size markers (M) are shown.

Figure 12

HMGB1 Stimulates CAG Repeat Expansion mediated by APE1 (from Figure 7 of ref. 48). The effect of HMGB1 on the CAG repeat expansion mediated by APE1 was examined under limiting concentration of APE1 (0.1 nM).

Figure 13

HMGB1 Stimulates APE1 Incision Activity in the Context of CAG Repeat. APE1 (0.1 nM) incision activity on a synthesized abasic site (THF) imbedded in CAG repeats is significantly increased by increasing concentrations of HMGB1 (Lane 2–5). Lane 1 represents APE1 (0.1 nM) incision in the absence of HMGB1. Lane 2–5 represents APE1 incision in the presence of 10, 20, 50 and 100 nM HMGB1, respectively. Substrate and product are indicated by arrows. The quantification of APE1 incision product is illustrated in lower panel.

Figure 14

HMGB1 Binds to CAG Repeat Hairpins. The binding ability of HMGB1 to various size of CAG repeat hairpin was examined by gel mobility shift assay. HMGB1 binding to (CAG)₁, (CAG)₂ and (CAG)₅ was poor. However, its binding to (CAG)₁₂ was significantly enhanced with its increasing concentrations (Lane 2–4) suggesting that HMGB1 prefers to bind to larger CAG repeat hairpin. Lane 1 represents binding mixture without HMGB1. Lane 2–4 represents binding mixture containing 20, 50, 100 and 200 nM HMGB1, respectively. DNA substrate and HMGB1-DNA complex are indicated by arrows.