Glyceraldehyde-3-phosphate dehydrogenase is required for efficient repair of cytotoxic DNA lesions in Escherichia coli

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response.     

Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA

Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation, and benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so, and likely modulates their repair by the NER machinery. The abundance of HMGB1 suggests that it may play an important role in determining the sensitivity of cells to DNA damage under physiological, experimental, and therapeutic conditions.     

HMGB1 protein inhibits DNA replication in vitro: a role of the acetylation and the acidic tail

The high mobility group box (HMGB) 1 protein is a very abundant and conserved protein that is implicated in many key cellular events but its functions within the nucleus remain elusive. The role of this protein in replication of closed circular DNA containing a eukaryotic origin of replication has been studied in vitro by using native and recombinant HMGB1 as well as various modified HMGB1 preparations such as truncated protein, lacking its C-terminal tail, in vivo acetylated protein, and recombinant HMGB1 phosphorylated in vitro by protein kinase C (PKC). Native HMGB1 extracted from tumour cells inhibits replication and this effect is reduced upon acetylation and completely abolished upon removal of the acidic C-terminal tail. Recombinant HMGB1, however, fails to inhibit replication but it acquires such a property following in vitro phosphorylation by PKC.     

Role of the glycolytic protein,glyceraldehyde-3-phosphate dehydrogenase,in normal cell function and in cell pathology

The glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be an archtypical protein of limited excitement. However, independent studies from a number of different laboratories reported a variety of diverse biological properties of the GAPDH protein. As a membrane protein, GAPDH functions in endocytosis; in the cytoplasm, it is involved in the translational control of gene expression; in the nucleus, it functions in nuclear tRNA export, in DNA replication, and in DNA repair. The intracellular localization of GAPDH may be dependent on the proliferative state of the cell. Recent studies identified a role for GAPDH in neuronal apoptosis. GAPDH gene expression was specifically increased during programmed neuronal cell death. Transfection of neuronal cells with antisense GAPDH sequences inhibited apoptosis. Lastly, GAPDH may be directly involved in the cellular phenotype of human neurodegenerative disorders, especially those characterized at the molecular level by the expansion of CAG repeats. In this review, the current status of ongoing GAPDH studies are described (with the exception of its unique oxidative modification by nitric oxide). Consideration of future directions are suggested. J. Cell. Biochem. 66:133-140, 1997. © 1997 Wiley-Liss, Inc.     

The Structural Organization of the HMGB1 Nuclear Protein and Its Effect on the Formation of Ordered Supramolecular Complexes

Biophysics - HMGB1 is one of the key proteins of the cell. HMGB1 performs its main functions predominantly in the cell nucleus, as an essential component of DNA–protein and multiprotein...     

HMGB1 interacts with XPA to facilitate the processing of DNA interstrand crosslinks in human cells

Many effective agents used in cancer chemotherapy cause DNA interstrand crosslinks (ICLs), which covalently link both strands of the double helix together resulting in cytotoxicity. ICLs are thought to be processed by proteins from a variety of DNA repair pathways; however, a clear understanding of ICL recognition and repair processing in human cells is lacking. Previously, we found that the high mobility group box 1 (HMGB1) protein bound to triplex-directed psoralen ICLs (TFO-ICLs) in vitro, cooperatively with NER damage recognition proteins, promoted removal of UVC-induced lesions and facilitated error-free repair of TFO-ICLs in mouse fibroblasts. Here, we demonstrate that HMGB1 recognizes TFO-ICLs in human cells, and its depletion increases ICL-induced mutagenesis in human cells without altering the mutation spectra. In contrast, HMGB1 depletion in XPA-deficient human cells significantly altered the ICL-induced mutation spectrum from predominantly T→A to T→G transversions. Moreover, the recruitment of XPA and HMGB1 to the ICLs is co-dependent. Finally, we show that HMGB1 specifically introduces negative supercoils in ICL-containing plasmids in HeLa cell extracts. Taken together, our data suggest that in human cells, HMGB1 functions in association with XPA on ICLs and facilitates the formation of a favorable architectural environment for ICL repair processing.     

Determinants of specific binding of HMGB1 protein to hemicatenated DNA loops

Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA-protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA).poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd approximately 0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell.     

Native HMGB1 protein inhibits repair of cisplatin-damaged nucleosomes in vitro

The high mobility group box (HMGB) 1 protein, one of the most abundant nuclear non-histone proteins has been known for its inhibitory effect on repair of DNA damaged by the antitumor drug cisplatin. Here, we report the first results that link HMGB1 to repair of cisplatin-treated DNA at nucleosome level. Experiments were carried out with three types of reconstituted nucleosomes strongly positioned on the damaged DNA: linker DNA containing nucleosomes (centrally and end-positioned) and core particles. The highest repair synthesis was registered with end-positioned nucleosomes, two and three times more efficient than that with centrally positioned nucleosomes and core particles, respectively. HMGB1 inhibited repair of linker DNA containing nucleosomes more efficiently than that of core particles. Just the opposite was the effect of the in vivo acetylated HMGB1: stronger repair inhibition was obtained with core particles. No inhibition was observed with HMGB1 lacking the acidic tail. Binding of HMGB1 proteins to different nucleosomes was also analysed. HMGB1 bound preferentially to damage nucleosomes containing linker DNA, while the binding of the acetylated protein was linker independent. We show that both the repair of cisplatin-damaged nucleosomes and its inhibition by HMGB1 are nucleosome position-dependent events which are accomplished via the acidic tail and modulated by acetylation.     

HMGB1: endogenous danger signaling

While foreign pathogens and their products have long been known to activate the innate immune system, the recent recognition of a group of endogenous molecules that serve a similar function has provided a framework for understanding the overlap between the inflammatory responses activated by pathogens and injury. These endogenous molecules, termed alarmins, are normal cell constituents that can be released into the extracellular milieu during states of cellular stress or damage and subsequently activate the immune system. One nuclear protein, High mobility group box-1 (HMGB1), has received particular attention as fulfilling the functions of an alarmin by being involved in both infectious and non-infectious inflammatory conditions. Once released, HMGB1 signals through various receptors to activate immune cells involved in the immune process. Although initial studies demonstrated HMGB1 as a late mediator of sepsis, recent findings indicate HMGB1 to have an important role in models of non-infectious inflammation, such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. Furthermore, in contrast to its pro-inflammatory functions, there is evidence that HMGB1 also has restorative effects leading to tissue repair and regeneration. The complex functions of HMGB1 as an archetypical alarmin are outlined here to review our current understanding of a molecule that holds the potential for treatment in many important human conditions.     

Evidence for involvement of HMGB1 protein in human DNA mismatch repair

Defects in human DNA mismatch repair predispose to cancer, but many components of the pathway have not been identified. We report here the identification and characterization of a novel component required for mismatch repair in human cells. A 30-kDa protein was purified to homogeneity by virtue of its ability to complement a depleted HeLa extract in repair of mismatched heteroduplexes. The complementing activity was identified as HMGB1 (the high mobility group box 1 protein), a non-histone chromatin protein that facilitates protein-protein interactions and recognizes DNA damage. Evidence is also presented that HMGB1 physically interacts with MutSalpha and is required at a step prior to the excision of mispaired nucleotide in mismatch repair.     

Sequence-specific recognition of cancer drug-DNA adducts by HMGB1a repair protein

The efficacy of cancer drugs such as cisplatin (Cp) and oxaliplatin (Ox), which covalently bind to DNA to form drug-DNA adducts, is linked to their recognition by repair proteins such as HMGB1a. Previous experimental studies showed that HMGB1a's binding affinity for Cp- and Ox-DNA varies with the drug used and the local DNA sequence context of the adduct. We link this differential binding affinity to the free energy of deforming (bending and minor groove opening) the drug-DNA molecule during HMGB1a binding. Specifically, the minimal binding affinity of HMGB1a for Ox-DNA in the TGGA context is explained by its larger deformation free energy compared with Cp-DNA or Ox-DNA in other sequence contexts. Methyl groups on neighboring thymine bases in Ox-TGGA crowd the minor groove and sterically hinder the motion of the diaminocyclohexane ring of Ox, leading to this reduced deformability and resultant decrease in HMGB1a's binding affinity.