High-affinity DNA binding of HU protein from the hyperthermophile Thermotoga maritima 1 1Edited by T. Richmond

Grove, Anne; Lim, Lynette

doi:10.1006/jmbi.2001.4763

Cited by 34 publications

(83 citation statements)

References 54 publications

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“…The Size of the Dps-1 Binding Site-The affinity of Dps-1 for duplex DNA of decreasing length was measured to evaluate the binding site size of Dps-1 (32). Consistent with the previously reported ϳ0.5 nM affinity for 26-bp DNA (24), quantification of complex formation with 26-bp DNA yields a K d of 0.40 Ϯ 0.04 nM for dodecameric Dps-1 (Fig.…”

Section: Iron Oxidation Is Not Compromised In Dps-dn and Dps-met-supporting

confidence: 77%

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The N-terminal Extensions of Deinococcus radiodurans Dps-1 Mediate DNA Major Groove Interactions as well as Assembly of the Dodecamer

Bhattacharyya

Grove

2007

Journal of Biological Chemistry

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Dps (DNA protection during starvation) proteins play an important role in the protection of prokaryotic macromolecules from damage by reactive oxygen species. Previous studies have suggested that the lysine-rich N-terminal tail of Dps proteins participates in DNA binding. In comparison with other Dps proteins, Dps-1 from Deinococcus radiodurans has an extended N terminus comprising 55 amino acids preceding the first helix of the 4-helix bundle monomer. In the crystal structure of Dps-1, the first ϳ30 N-terminal residues are invisible, and the remaining 25 residues form a loop that harbors a novel metalbinding site. We show here that deletion of the flexible N-terminal tail obliterates DNA/Dps-1 interaction. Surprisingly, deletion of the entire N terminus also abolishes dodecameric assembly of the protein. Retention of the N-terminal metal site is necessary for formation of the dodecamer, and metal binding at this site facilitates oligomerization of the protein. Electrophoretic mobility shift assays using DNA modified with specific major/minor groove reagents further show that Dps-1 interacts through the DNA major groove. DNA cyclization assays suggest that dodecameric Dps-1 does not wrap DNA about itself. A significant decrease in DNA binding affinity accompanies a reduction in duplex length from 22 to 18 bp, but only for dodecameric Dps-1. Our data further suggest that high affinity DNA binding depends on occupancy of the N-terminal metal site. Taken together, the mode of DNA interaction by dodecameric Dps-1 suggests interaction of two metal-anchored N-terminal tails in successive DNA major grooves, leading to DNA compaction by formation of stacked protein-DNA layers.All aerobic microorganisms are exposed to reactive oxygen species ( OH ϩ Fe 3ϩ ) (1). Thus, the presence of iron increases the probability of oxidative damage to cellular components. Dps, initially studied in Escherichia coli, was shown to protect DNA by its ability to chelate ferrous iron and also by its physical association with DNA (2-5).Twelve Dps monomers form a spherical assembly similar to the spherical shell formed by 24 subunits of the iron storage protein, ferritin (6 -9). Each Dps monomer adopts a four-helix (A-D) bundle conformation as seen for ferritin, but unlike ferritin, Dps possesses a short helix in the middle of the BC loop and lacks the C-terminal fifth helix present in the ferritin monomer (10 -13). Secondly, the ferroxidase site in Dps is usually generated at the interface between two subunits and, with one reported exception, is not within the four-helix bundle as in the case of ferritin (14,15). It is also notable that not all Dps homologs follow the same catalytic mechanism, as exemplified by the absence of a conserved ferroxidase center in Lactococcus lactis DpsB and the failure of Bacillus anthracis Dps1 to utilize H 2 O 2 in the ferroxidation reaction (16,17).In contrast to the highly conserved ferroxidase center, Dps homologs have a variable N-terminal extension. This N-terminal tail, which contains multiple positively ...

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Section: Iron Oxidation Is Not Compromised In Dps-dn and Dps-met-supporting

confidence: 77%

“…The sequence of 26-, 22-, 18-, and 13-bp (all average G ϩ C content) DNA is available as supplemental material. The top strand was 32 P-labeled at the 5Ј-end with phage T4 polynucleotide kinase. Equimolar amounts of complementary oligonucleotides were mixed, heated to 90°C, and cooled slowly to room temperature (23°C) to form duplex DNA.…”

Section: Methodsmentioning

confidence: 99%

The N-terminal Extensions of Deinococcus radiodurans Dps-1 Mediate DNA Major Groove Interactions as well as Assembly of the Dodecamer

Bhattacharyya

Grove

2007

Journal of Biological Chemistry

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“…However, loop formation and breakdown were observed for DNA unwound by 3% or more ( ϭ Ϫ0.03 or Ϫ0.06), which exceeds the 1.5% required to denature AT-rich regions in DNA under tension (14)(15)(16). Considering that the HU binding site at the apex of the gal DNA loop is AT rich and palindromic (9,17), and that HU preferentially binds to altered DNA structures such as denatured or cruciform DNA (18)(19)(20)(21)(22), we reasoned that HU may facilitate GalR-mediated looping by tightly binding the single-stranded DNA (ssDNA) in the unwound segment induced by negative supercoiling. Such a conformation would lower the energy required for looping, because ssDNA is more flexible than double-stranded DNA.…”

Section: Resultsmentioning

confidence: 99%

Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping

Lia

Bensimon

Croquette

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

104

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The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and͞or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of Ϸ40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR͞HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR͞HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination. T he nucleoid structure in the bacterium Escherichia coli contains a circular DNA molecule of 4.7 million bp present in highly condensed form. The condensation is mediated by DNA supercoiling and the binding of several small nucleoidassociated proteins, e.g., heat unstable nucleoid protein (HU), integration host factor (IHF), factor for inversion stimulation (FIS), histone-like nucleoid structuring protein (HNS), suppressor of thymidylate synthase mutant phenotype A (StpA), and DNA binding protein from starved cells (Dps). These proteins are known to bend DNA or bind to altered structures of DNA. It is suggested that these proteins are mainly responsible for the compaction of DNA in a way that distinguishes the bacterial nucleoid from eukaryotic chromatin. These proteins are also associated with the machinery of macromolecular biosynthesis, including RNA polymerase and specific gene-regulatory, DNAbinding proteins such as repressors and activators. Indeed, DNA may serve as a scaffold for the organized recruitment and assembly of proteins at specific positions to create nucleoprotein complexes with specific activity and regulatory properties. Such positioning has long been postulated to be the mechanism of repression of the gal operon by the gal repressor dimer protein (GalR). GalR represses transcription initiation from the two promoters, P1 and P2, of the gal operon by binding to two spatially separated operators, O E and O I , which encompass the promoters (1). Repression also requires supercoiled DNA and the presence of the nucleoid-associated protein HU (2). It has been proposed that a DNA loop generated by the interaction of the two operator-bound gal repressors inactivates the promoter (3). Repression of the gal operon would, th...

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“…DNA minicircles were generated by intramolecular ligation of the 32 P-labeled 105-bp DNA fragment with 20 units of T4 DNA ligase in the presence of Thermotoga maritima HU for 2 h at room temperature (34). Samples were treated with Exonuclease III for 1 h at room temperature and the reactions quenched with stop buffer.…”

Section: Methodsmentioning

confidence: 99%

The Saccharomyces cerevisiae High Mobility Group Box Protein HMO1 Contains Two Functional DNA Binding Domains

Kamau

Bauerle

Grove

2004

Journal of Biological Chemistry

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High mobility group box (HMGB) proteins are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as 4-way junctions. HMO1 is one of 10 Saccharomyces cerevisiae HMGB proteins, and it is required for normal growth and plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease. Using electrophoretic mobility shift assays, we have shown here that HMO1 binds 26-bp duplex DNA with K d ‫؍‬ 39.6 ؎ 5.0 nM and that its divergent box A domain participates in DNA interactions, albeit with low affinity. HMO1 has only modest preference for DNA with altered conformations, including DNA with nicks, gaps, overhangs, or loops, as well as for 4-way junction structures and supercoiled DNA. HMO1 binds 4-way junctions with half-maximal saturation of 19.6 ؎ 2.2 nM, with only a modest increase in affinity in the absence of magnesium ions (half-maximal saturation 6.1 ؎ 1.1 nM). Whereas the box A domain contributes modest structure-specific binding, the box B domain is required for high affinity binding. HMO1 bends DNA, as measured by DNA cyclization assays, facilitating cyclization of 136-, 105-, and 87-bp DNA, but not 75-bp DNA, and it has a significantly longer residence time on DNA minicircles compared with linear duplex DNA. The unique DNA binding properties of HMO1 are consistent with global roles in the maintenance of chromatin structure. High mobility group (HMG)1 proteins constitute a significant proportion of non-histone proteins of eukaryotic chromatin. They are abundant proteins that are grouped, in part based on their DNA binding characteristics, into three major classes, HMGA, HMGB, and HMGN (1-4). HMGB proteins contain one or more homologous repeats of the ϳ80-amino acid sequence HMG box and are classified into two families based on the abundance, function, and DNA specificity of this conserved region (1, 5, 6). The moderately sequence-specific family is typified by transcription factors such as sex-determining factor SRY and lymphoid enhancer factor LEF-1 (7, 8), whereas the non-sequence-specific family is represented by so-called architectural factors HMGB1/2 and the Saccharomyces cerevisiae non-histone chromosomal proteins 6A and 6B (NHP6A/B) (9).The tertiary structures of HMG boxes from sequence-specific and non-sequence-specific proteins have revealed an evolutionarily conserved, common global fold consisting of an L-shaped structure composed of three ␣-helices (10 -19). The HMG DNA binding domain, which interacts with ϳ10 bp of duplex, binds to the minor groove of DNA by partial intercalation of one or two surface-exposed, conserved hydrophobic residues into the base pair stack. Consequently, the DNA is greatly distorted, resulting in a sharp bend and helical underwinding (4,8,14,16,20). SRY and LEF-1 cause bending by insertion of helix I hydrophobic residues Ile and Met, respectively, into the base pair stack (Fig. 1) (8, 17). HMGB1 contains tandem HMG box domains referred to as box A and box B; DNA-intercalating residues are located...

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High-affinity DNA binding of HU protein from the hyperthermophile Thermotoga maritima 1 1Edited by T. Richmond

Cited by 34 publications

References 54 publications

The N-terminal Extensions of Deinococcus radiodurans Dps-1 Mediate DNA Major Groove Interactions as well as Assembly of the Dodecamer

The N-terminal Extensions of Deinococcus radiodurans Dps-1 Mediate DNA Major Groove Interactions as well as Assembly of the Dodecamer

Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping

The Saccharomyces cerevisiae High Mobility Group Box Protein HMO1 Contains Two Functional DNA Binding Domains

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