The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds. We describe the characterization of HucR, a novel MarR homolog from Deinococcus radiodurans that demonstrates phenolic sensing capabilities. HucR binds as a homodimer to a single site within its promoter/operator region with K d ‫؍‬ 0.29 ؎ 0.02 nM. The HucR binding site contains a pseudopalindromic sequence, composed of 8-bp half-sites separated by 2 bp. The location of the HucR binding site in the intergenic region between hucR and a putative uricase suggests a mechanism of simultaneous co-repression of these two genes. The substrate of uricase, uric acid, is an efficient antagonist of DNA binding, reducing HucR-DNA complex formation to 50% at 0.26 mM ligand, compared with 5.2 and 46 mM for the aromatic compounds salicylate and acetylsalicylate, respectively. Enhanced levels in vivo of hucR and uricase transcript and increased uricase activity under conditions of excess uric acid further indicate a novel regulatory mechanism of aromatic catabolism in D. radiodurans. Since uric acid is a scavenger of reactive oxygen species, we hypothesize that HucR is a participant in the intrinsic resistance of D. radiodurans to high levels of oxidative stress.

Bacterial iron storage proteins such as ferritin serve as intracellular iron reserves. Members of the DNA protection during starvation (Dps) family of proteins are structurally related to ferritins, and their function is to protect the genome from iron-induced free radical damage. Some members of the Dps family bind DNA and are thought to do so only as fully assembled dodecamers. We present the cloning and characterization of a Dps homolog encoded by the radiation-resistant eubacterium Deinococcus radiodurans and show that DNA binding does not require its assembly into a dodecamer. D.radiodurans Dps-1, the product of gene DR2263, adopts a stably folded conformation, as demonstrated by circular dichroism spectroscopy, and undergoes a transition to a disordered state with a melting temperature of 69.2(+/-0.1) degrees C. While a dimeric form of Dps-1 is observed under low-salt conditions, a dodecameric assembly is highly favored at higher concentrations of salt. Both oligomeric forms of Dps-1 exhibit ferroxidase activity, and Fe(II) oxidation/mineralization is seen for dodecameric Dps-1. Notably, addition of Ca(2+) (to millimolar concentrations) to dodecameric Dps-1 can result in the reduction of bound Fe(III). Dimeric Dps-1 protects DNA from both hydroxyl radical cleavage and from DNase I-mediated cleavage; however, dodecameric Dps-1 is unable to provide efficient protection against hydroxyl radical-mediated DNA cleavage. While dodecameric Dps-1 does bind DNA, resulting in formation of large aggregates, cooperative DNA binding by dimeric Dps-1 leads to formation of protein-DNA complexes of finite stoichiometry.

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We report here the 2.3 A resolution structure of the hypothetical uricase regulator (HucR) from Deinococcus radiodurans R1. HucR, a member of the MarR family of DNA-binding proteins, was previously shown to repress its own expression as well as that of a uricase, a repression that is alleviated both in vivo and in vitro upon binding uric acid, the substrate for uricase. As uric acid is a potent scavenger of reactive oxygen species, and as D. radiodurans is known for its remarkable resistance to DNA-damaging agents, these observations indicate a novel oxidative stress response mechanism. The crystal structure of HucR in the absence of ligand or DNA reveals a dimer in which the DNA recognition helices are preconfigured for DNA binding. This configuration of DNA-binding domains is achieved through an apparently stable dimer interface that, in contrast to what is observed in other MarR homologs for which structures have been determined, shows little conformational heterogeneity in the absence of ligand. An additional amino-terminal segment, absent from other MarR homologs, appears to brace the principal helix of the dimerization interface. However, although HucR is preconfigured for DNA binding, the presence of a stacked pair of symmetry-related histidine residues at a central pivot point in the dimer interface suggests a mechanism for a conformational change to attenuate DNA binding.

SUMMARY1. The concentration of total protein in c.s.f. and plasma has been measured in fetal sheep of different gestational ages and in the adult. In c.s.f. it was highest (approximately 840 mg/100 ml.) in the youngest fetuses (35 days) and declined steeply by 60 days (260 mg/tOO ml.). It decreased less markedly in the last half of gestation to reach about 50 mg/100 ml. at 125 days which is twice the adult value. Protein concentration in plasma was lowest in the youngest fetuses and did not rise much until the second half of gestation during which time it doubled. There was a further rather larger increase between the late fetal (125 days) stage and the adult.2. Albumin, fetuin, a-fetoprotein (AFP), transferrin and lipoprotein were identified in c.s.f. and plasma.3. The concentrations of albumin, AFP and fetuin in c.s.f. and plasma at different gestational ages were measured using immunodiffusion and radioimmuno-assays.4. Albumin was the major protein in plasma at all ages studied (35-128 days gestation and adult). Its concentration increased throughout gestation whereas that of fetuin was similar at all fetal ages and that of AFP declined markedly during the second half of gestation. In the adult, fetuin was only about 0.1 % of the total protein in plasma and AFP was not detectable.5. In 35-40 day fetuses albumin, AFP and fetuin accounted for 90 % of the total protein concentration in plasma and for about 70-80 % of the total protein concentration of c.s.f. As gestation progressed the concentration of all three proteins in c.s.f. declined. But the concentration of AFP and fetuin fell more rapidly and to a greater extent than that of albumin; neither AFP nor fetuin could be detected in adult c.s.f.6. The c.s.f.: plasma ratio was above 50 % for AFP and above 60 % for fetuin at 35 days compared with about 25 % for albumin at the same fetal age. The c.s.f.: plasma ratios for all three proteins declined with increasing fetal age and were not significantly different from each other by about mid gestation.

Biopsy tissues were fixed in buffered formalin and processed routinely through paraffin wax, ensuring optimal orientation at the embedding stage. Sections (5 [t) were cut at six levels and stained with haematoxylin and eosin, periodic acid Schiff, and high iron diamine alcian blue (HIDAB), the latter with strict pH con-

Members of the MarR family of winged helix transcriptional regulators have been shown to regulate multidrug and oxidative stress response, pathogenesis, and catabolism of aromatic compounds. Many respond to anionic lipophilic compounds in their capacity to bind DNA, and the co-crystal structure of MarR bound to salicylate revealed two ligand-binding pockets, SAL-A and SAL-B. The MarR homolog, HucR, from Deinococcus radiodurans has been shown to repress expression of a predicted uricase, and DNA-binding by HucR is antagonized by uric acid, the substrate of uricase. We provide a biochemical investigation of DNA-binding and uric acid-binding by HucR. Equilibrium analytical ultracentrifugation indicates that HucR exists as a dimer. Intrinsic fluorescence spectra suggest that the association of the HucR dimer with its cognate DNA involves conformational flexibility in the globular interior and/or dimerization domain of the protein, and near-UV circular dichroism spectra indicate a concomitant change in the helical twist of the DNA duplex. DNA-binding affinity, measured by electrophoretic mobility-shift assays, for HucR mutants bearing single amino acid substitutions suggests the importance of the beta-hairpin "wing" in DNA binding. Analysis of intrinsic fluorescence spectra demonstrates that uric acid induces conformational changes in HucR and binds with an apparent K(d)=11.6(+/-3.7)muM and a Hill coefficient of 0.7+/-0.1, indicating negative cooperativity. Fluorescence and DNA-binding properties of the HucR variants indicate that SAL-A is a low-affinity, uric acid-binding site and that negative cooperativity exists between homologous, high-affinity sites. The conservation of residues comprising site SAL-A suggests that it is a low-affinity, ligand-binding site in MarR homologs. Mechanistic considerations suggest that HucR is regulated by uric acid to maintain optimal cellular levels of this scavenger of free radicals in response to oxidative stress and DNA damage.

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