High mobility group (HMG) proteins: Modulators of chromatin structure and DNA repair in mammalian cells

It has been almost a decade since the last review appeared comparing and contrasting the influences that the different families of High Mobility Group proteins (HMGA, HMGB and HMGN) have on the various DNA repair pathways in mammalian cells. During that time considerable progress has been made in our understanding of how these non-histone proteins modulate the efficiency of DNA repair by all of the major cellular pathways: nucleotide excision repair, base excision repair, double-stand break repair and mismatch repair. Although there are often similar and over-lapping biological activities shared by all HMG proteins, members of each of the different families appear to have a somewhat ‘individualistic’ impact on various DNA repair pathways. This review will focus on what is currently known about the roles that different HMG proteins play in DNA repair processes and discuss possible future research areas in this rapidly evolving field.     

高迁移率族蛋白

高迁移率族蛋白(highmobilitygroupprotein,HMG蛋白)广泛存在于真核生物细胞中,因其在聚丙稀凝胶电泳中的高迁移率而得名。HMG蛋白是真核细胞基因调控的动力体现者,是真核细胞内继组蛋白之后含量最为丰富的一组染色质蛋白质,它们在染色质的结构与功能及基因表达调控过程中均发挥着重要作用。HMG蛋白家族可分为HMGA、HMGB和HMGN三类亚家族。现对HMG蛋白家族的三类亚家族蛋白HMGA、HMGB和HMGN的结构与功能进行综述。     

High mobility group proteins and their post-translational modifications

The high mobility group (HMG) proteins, including HMGA, HMGB and HMGN, are abundant and ubiquitous nuclear proteins that bind to DNA, nucleosome and other multi-protein complexes in a dynamic and reversible fashion to regulate DNA processing in the context of chromatin. All HMG proteins, like histone proteins, are subjected to extensive post-translational modifications (PTMs), such as lysine acetylation, arginine/lysine methylation and serine/threonine phosphorylation, to modulate their interactions with DNA and other proteins. There is a growing appreciation for the complex relationship between the PTMs of HMG proteins and their diverse biological activities. Here, we reviewed the identified covalent modifications of HMG proteins, and highlighted how these PTMs affect the functions of HMG proteins in a variety of cellular processes.     

高迁移率族蛋白与真核基因表达调控

高迁移率族蛋白 (high mobility group protein , HMG) 是一系列的染色质相关蛋白,广泛存在于真核生物细胞中,含量丰富,因其在聚丙烯酰胺凝胶电泳中的高迁移率而得名 . HMG 蛋白家族可分为 HMGB 、 HMGA 和 HMGN 三类亚家族,各亚家族有其特征的结构域,这些结构域介导了 HMG 和 DNA 或染色质相关区域的相互作用 . 现已发现这些蛋白质具有多种重要生物学功能,其中几乎所有 HMG 都可以通过修饰、弯曲或改变染色质 /DNA 的结构,促进各种蛋白质因子形成大分子复合物来调节基因转录 .     

Interaction of maize chromatin-associated HMG proteins with mononucleosomes: role of core and linker histones

Two groups of plant chromatin-associated high mobility group (HMG) proteins, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain, have been identified. We have examined the interaction of recombinant maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA in the presence of H1 differed from that observed in the absence of H1. HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity. The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones), suggesting that the histone N-termini are dispensable for HMG protein binding. In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main interaction partner of HMGB1 rather than the histone N-termini. In conclusion, these results indicate that specific nucleosome binding of the plant HMGB proteins requires simultaneous DNA and histone contacts.     

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.     

Overlapping expression patterns among the genes encoding Arabidopsis chromosomal high mobility group (HMG) proteins

High mobility group (HMG) proteins are usually considered ubiquitous components of the eukaryotic chromatin. Using HMG gene promoter-GUS reporter gene fusions we have examined the expression of the reporter gene in transgenic Arabidopsis plants. These experiments have revealed that the different HMGA and HMGB promoters display overlapping patterns of activity, but they also show tissue- and developmental stage-specific differences. Moreover, leader introns that are present in some of the HMGB genes can modulate reporter gene expression. The differential HMG gene expression supports the view that the various HMG proteins serve partially different architectural functions in plant chromatin.     

Transient HMGB protein interactions with B-DNA duplexes and complexes

HMGB proteins are abundant, non-histone proteins in eukaryotic chromatin. HMGB proteins contain one or two conserved “HMG boxes” and can be sequence-specific or nonspecific in their DNA binding. HMGB proteins cause strong DNA bending and bind preferentially to deformed DNAs. We wish to understand how HMGB proteins increase the apparent flexibility of non-distorted B-form DNA. We test the hypothesis that HMGB proteins bind transiently, creating an ensemble of distorted DNAs with rapidly interconverting conformations. We show that binding of B-form DNA by HMGB proteins is both weak and transient under conditions where DNA cyclization is strongly enhanced. We also detect novel complexes in which HMGB proteins simultaneously bind more than one DNA duplex.     

HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.     

Interaction of wheat high-mobility-group proteins with four-way-junction DNA and characterization of the structure and expression of HMGA gene

Plant high-mobility-group (HMG) chromosomal proteins are the most abundant and ubiquitous nonhistone proteins found in the nuclei of higher eukaryotes. There are only two families of HMG proteins, namely, HMGA and HMGB in plants. The cDNA encoding wheat HMGa protein was isolated and characterized. Wheat HMGA cDNA encodes a protein of 189 amino acid residues. At its N terminus, there is a histone H1-like structure, which is a common feature of plant HMGA proteins, followed by four AT-hook motifs. Polymerase chain reaction results show that the gene contains a single intron of 134 bp. All four AT-hook motifs are encoded by the second exon. Northern blot results show that the expression of HMGA gene is much higher in organs undergoing active cell proliferation. Gel retardation analysis show that wheat HMGa, b, c and histone H1 bind to four-way-junction DNA with high binding affinity, but affinity is dramatically reduced with increasing Mg(2+) and Na(+) ion concentration. Competition binding studies show that proteins share overlapping binding sites on four-way-junction DNA. HMGd does not bind to four-way-junction DNA.     

Protein kinase CK2 differentially phosphorylates maize chromosomal high mobility group B (HMGB) proteins modulating their stability and DNA interactions.

The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins.