Mutational analysis of the DNA binding domain A of chromosomal protein HMG1

Falciola, Luca; Murchie, Alastair I. H.; Lilley, David M. J.; Bianchi, Marco E.

doi:10.1093/nar/22.3.285

Cited by 52 publications

(47 citation statements)

References 21 publications

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“…1, marked with stars) as in HMG1 domain B, suggesting that the Sin1p-(224 -304) peptide might have a rigid structure as well. It was shown that a W45R mutation in HMG1 A domain reduced the ability of this domain to bind 4WJDNA because of disruption of an ␣-helix (34,35). We, therefore, first asked how an analogous mutation in GST-Sin1p-(224 -304), W267R, might affect its binding to 4WJDNA.…”

Section: Sin1p/spt2p Domains Bind 4wjdnamentioning

confidence: 99%

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Functional Domains of the Yeast Chromatin Protein Sin1p/Spt2p Can Bind Four-way Junction and Crossing DNA Structures

Novoseler

Hershkovits

Katcoff

2005

Journal of Biological Chemistry

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Sin1p/Spt2p is a yeast chromatin protein that, when mutated or deleted, alters the transcription of a family of genes presumably by modulating local chromatin structure. In this study, we investigated the ability of different domains of this protein to bind four-way junction DNA (4WJDNA) since 4WJDNA can serve as a model for bent double helical DNA and for the crossed structure formed at the exit and entry of DNA to the nucleosomes. Sequence alignment of Sin1p/Spt2p homologues from 11 different yeast species showed conservation of several domains. We found that three domains of Sin1p/ Spt2p fused to glutathione S-transferase can each bind independently in a structure-specific manner to 4WJDNA as measured in a gel mobility shift assay. A feature common to these domains is a cluster of positively charged amino acids. Modification of this cluster resulted in either abolishment of binding or a change in the binding properties. One of the domains tested clearly bound superhelical DNA, although it failed to induce bending in a circularization assay. Poly-L-lysine, which may be viewed as a cluster of positively charged amino acids, bound 4WJDNA as well. Phenotypic analysis showed that disruption of any of these domains resulted in suppression of a his4-912␦ allele, indicating that each domain has functional significance. We propose that Sin1p/Spt2p is likely to modulate local chromatin structure by binding two strands of double-stranded DNA at their crossover point.Sin1p/Spt2p is a yeast chromatin non-histone protein. While the precise function of the protein is still unknown, it is known to function as a negative transcriptional regulator of a number of genes including SUC2 (1), INO1 (2), and SSA3 (3). Activity of the HO promoter, as measured from an HO promoter driving a lacZ gene (4), is also regulated by SIN1/SPT2. swi1, swi2, and swi3 mutants are unable to transcribe RNA from the HO promoter. However, in sin1/swi double mutants, transcription is restored. Mutants in SPT2 were first identified as suppressors of ty and ␦ insertions in the 5Ј non-coding region of the HIS4 gene (5). As the negative regulation of these genes is overcome by the SW1/SNF chromatin remodeling complex, it was suggested that a function of SIN1p/SPT2p is to somehow maintain chromatin compaction at specific locations in the chromatin.

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Section: Sin1p/spt2p Domains Bind 4wjdnamentioning

confidence: 99%

“…Mammalian HMG1 box A contains the sequence KKPRGKM. A single point mutation replacing the arginine with glycine reduces the affinity of this protein for 4WJDNA at least 10-fold (34).…”

Section: Sin1p/spt2p Domains Bind 4wjdnamentioning

confidence: 99%

Functional Domains of the Yeast Chromatin Protein Sin1p/Spt2p Can Bind Four-way Junction and Crossing DNA Structures

Novoseler

Hershkovits

Katcoff

2005

Journal of Biological Chemistry

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“…HMG1 and -2 are ubiquitous, abundantly expressed nuclear proteins that bind nonspecifically in the minor groove of DNA via their two HMG box domains. They bind preferentially to distorted DNA, including cruciform four-way junction DNA structures (4,13,30,40), and play a role in the repair of 1,2-cis-platinum DNA intrastrand adducts (39). HMG1 and -2 also bind to and preserve negatively supercoiled DNA structures (48).…”

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confidence: 99%

Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination

Ciubotaru

Schatz

2004

Molecular and Cellular Biology

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V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.The process of immunoglobulin and T-cell receptor gene rearrangement, known as V(D)J recombination, occurs during lymphocyte differentiation and plays a major role in diversifying these antigen receptors in jawed vertebrates (55). Two steps precede DNA double-strand cleavage by the RAG1/ RAG2 recombinase machinery. First, RAG1-RAG2 specifically bind to short DNA elements termed recombination signal sequences (RSSs) flanking the V, D, and J gene segments. RSSs are composed of two well-conserved sequences, a palindromic heptamer (consensus 5Ј-CACAGTG) and an A/T-rich nonamer (consensus 5Ј-ACAAAAACC) separated by a lesswell-conserved spacer region of 12 or 23 bp in length (41). The second step, the synapsis of a pair of RSSs, determines both the identity and the type of gene segments to be adjoined. It is within this paired complex that DNA double-strand breaks are introduced between the heptamer and flanking coding region (referred to as hairpin formation, because cleavage generates a covalently sealed hairpin structure at the end of each coding segment).In vivo, recombination occurs almost exclusively between gene segments flanked by a 12-and a 23-RSS (complementary RSSs) according to the 12/23 rule. The mechanisms enforcing this important restriction remain poorly understood at a molecular level. Biochemical experiments have revealed a more stringent adherence to the 12/23 rule in coupled cleavage assays (11,56,58) than in electrophoretic mobility shift assays that ass...

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“…Complexes of the [R6A]cHMGla with DNA exhibited reduced stability, as judged from the smearing of the shifted bands in the binding assay. A similar mutation in this region of ArglO of the HMGl-BD of the rat HMGl protein led to an approximately tenfold reduction of the binding affinity to four-way-junction DNA [28].…”

Section: Discussionmentioning

confidence: 94%

“…Unexpectedly, only moderate alterations of the DNAbinding and DNA-bending properties were observed in this mutant protein. Two previous mutational analyzes of the HMGlBDs of the rat HMGl protein have revealed that substitution of Trp42 in the central hydrophobic core similarly changed the conformational stability [28] and the structure [29] of the protein whereas the binding of the mutant protein to DNA was only slightly affected. The deletion of the first seven amino acid residues reduced the strength of the interaction of the cHMGla protein with DNA.…”

Section: Discussionmentioning

confidence: 94%

Structural and Functional Consequences of Mutations within the Hydrophobic Cores of the HMG1‐Box Domain of the Chironomus High‐Mobility‐Group Protein 1a

Wiśniewski

Hessler

Claus

et al. 1997

European Journal of Biochemistry

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The high-mobility-group protein 1 box domain (HMGI-BD) is a structural element found in several DNA-binding proteins in eukaryotic cells. Its structure is dominated by three a-helices. The spatial arrangement of these helices into an L-shaped molecule is maintained by a number of apolar residues organized into a main and a secondary hydrophobic core. To analyze the significance of these residues for proper folding, conformational stability, and ability to bind and bend DNA, we have mutated the highly conserved Trpl4 of the Chironomus HMGl a protein and have synthesized a series of N-terminally truncated forms. The observed alterations in DNA-binding and DNA-bending characteristics were correlated with structural consequences, as revealed by CD spectroscopy, limited trypsin digestion, and transverse urea gradient gel electrophoresis. Mutation of the Trpl4 residue (Chironomus [W14A]HMGla) and deletion of the seven N-terminal residues, respectively, which are members of the main and the secondary core of Chironomus HMGla, both resulted in a substantial unfolding of the protein. Unexpectedly, these mutants still retained their ability to bind and bend DNA. Conformational analysis of wildtype cHMGla and [W14A]cHMGIa showed that the proteins unfold at 2-4 M urea. In contrast, their DNA complexes persisted even at 6-8 M of the denaturant. Multiple contacts between the HMGl-BD and the DNA are probably responsible for the unusual stability of the complexes.Keywords: high-mobility-group protein 1 (HMGI) ; high-mobility-group-protein-I-box domain; DNA binding; DNA bending; protein folding.The high-mobility-group-protein-1 -box domain (HMGI -BD) protein family comprises more than 100 known proteins interacting with DNA (reviewed in [l]). They can be roughly grouped into two subfamilies (reviewed in [2, 31). The first subfamily represents molecules that recognize specific DNA sequences and occurs usually in particular types of cells or specific stages of development. The abundant nonhistone chromosomal proteins in eukaryotic cells, the high-mobility-group proteins 1 and 2 (HMGI and 2), are the most prominent members of the vecond subfamily. They comprise proteins exhibiting binding preference for A+T-rich, bent, distorted, negatively supercoiled, cruciform DNAs, and do not possess a marked DNA-sequence specificity.The HMGl-BDs are L-shaped molecules 14-61 comprising three helices with angles of about 80 degrees between the antiparallel helices I and IT, and helix 111. Recently, modes of sequence-specific binding of HMG1-BDs of the human sex-deterCorrespondence to J. R. Wiiniewski, 111.

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Mutational analysis of the DNA binding domain A of chromosomal protein HMG1

Cited by 52 publications

References 21 publications

Functional Domains of the Yeast Chromatin Protein Sin1p/Spt2p Can Bind Four-way Junction and Crossing DNA Structures

Functional Domains of the Yeast Chromatin Protein Sin1p/Spt2p Can Bind Four-way Junction and Crossing DNA Structures

Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination

Structural and Functional Consequences of Mutations within the Hydrophobic Cores of the HMG1‐Box Domain of the Chironomus High‐Mobility‐Group Protein 1a

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