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ABSTRACT: RecA plays a key role in homologous recombination, the induction of the DNA damage response through LexA cleavage and the activity of error-prone polymerase in Escherichia coli. RecA interacts with multiple partners to achieve this pleiotropic role, but the structural location and sequence determinants involved in these multiple interactions remain mostly unknown. Here, in a first application to prokaryotes, Evolutionary Trace (ET) analysis identifies clusters of evolutionarily important surface amino acids involved in RecA functions. Some of these clusters match the known ATP binding, DNA binding, and RecA-RecA homo-dimerization sites, but others are novel. Mutation analysis at these sites disrupted either recombination or LexA cleavage. This highlights distinct functional sites specific for recombination and DNA damage response induction. Finally, our analysis reveals a composite site for LexA binding and cleavage, which is formed only on the active RecA filament. These new sites can provide new drug targets to modulate one or more RecA functions, with the potential to address the problem of evolution of antibiotic resistance at its root.
PLoS Genetics 09/2011; 7(9):e1002244. · 8.69 Impact Factor
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ABSTRACT: Desmosterolosis, a rare disorder of cholesterol biosynthesis, is caused by mutations in DHCR24, the gene encoding the enzyme 24-dehydrocholesterol reductase (DHCR24). To date, desmosterolosis has been described in only two patients. Here we report on a third patient with desmosterolosis who presented after delivery with relative macrocephaly, mild arthrogryposis, and dysmorphic facial features. Brain MRI revealed hydrocephalus, thickening of the tectum and massa intermedia, mildly effaced gyral pattern, underopercularization, and a thin corpus callosum. The diagnosis of desmosterolosis was established by detection of significant elevation of plasma desmosterol levels and reduced enzyme activity of DHCR24 upon expression of the patient's DHCR24 cDNA in yeast. The patient was found to be a compound heterozygote for c.281G>A (p.R94H) and c.1438G>A (p.E480K) mutations. Structural and evolutionary analyses showed that residue R94 resides at the flavin adenine dinucleotide (FAD) binding site and is strictly conserved throughout evolution, while residue E480 is less conserved, but the charge shift substitution is accompanied by drastic changes in the local protein environment of that residue. We compare the phenotype of our patient with previously reported cases.
American Journal of Medical Genetics Part A 07/2011; 155A(7):1597-604. · 2.39 Impact Factor
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Johannes Häberle,
Oleg A Shchelochkov,
Jing Wang, Panagiotis Katsonis,
Lynn Hall,
Sara Reiss,
Angela Eeds,
Alecia Willis,
Meeta Yadav,
Samantha Summar,
Olivier Lichtarge,
Vicente Rubio,
Lee-Jun Wong,
Marshall Summar
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ABSTRACT: Deficiency of carbamoyl phosphate synthetase I (CPSI) results in hyperammonemia ranging from neonatally lethal to environmentally induced adult-onset disease. Over 24 years, analysis of tissue and DNA samples from 205 unrelated individuals diagnosed with CPSI deficiency (CPSID) detected 192 unique CPS1 gene changes, of which 130 are reported here for the first time. Pooled with the already reported mutations, they constitute a total of 222 changes, including 136 missense, 15 nonsense, 50 changes of other types resulting in enzyme truncation, and 21 other changes causing in-frame alterations. Only ∼10% of the mutations recur in unrelated families, predominantly affecting CpG dinucleotides, further complicating the diagnosis because of the "private" nature of such mutations. Missense changes are unevenly distributed along the gene, highlighting the existence of CPSI regions having greater functional importance than other regions. We exploit the crystal structure of the CPSI allosteric domain to rationalize the effects of mutations affecting it. Comparative modeling is used to create a structural model for the remainder of the enzyme. Missense changes are found to directly correlate, respectively, with the one-residue evolutionary importance and inversely correlate with solvent accessibility of the mutated residue. This is the first large-scale report of CPS1 mutations spanning a wide variety of molecular defects highlighting important regions in this protein.
Human Mutation 11/2010; 32(6):579-89. · 5.69 Impact Factor
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ABSTRACT: Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a progressive neurodegenerative disorder associated with thymidine phosphorylase deficiency resulting in high levels of plasma thymidine and a characteristic clinical phenotype.
To investigate the molecular basis of MNGIE in a patient with a normal plasma thymidine level.
Clinical, neurophysiological, and histopathological examinations as well as molecular and genetic analyses.
Nerve and muscle center and genetic clinic. Patient A 42-year-old woman with clinical findings strongly suggestive for MNGIE.
Clinical description of the disease and its novel genetic cause.
Identification of mitochondrial DNA depletion in muscle samples (approximately 12% of the control mean content) prompted us to look for other causes of our patient's condition. Sequencing of genes associated with mitochondrial DNA depletion-POLG, PEO1, ANT1, SUCLG1, and SUCLA2-did not reveal deleterious mutations. Results of sequencing and array comparative genomic hybridization of the mitochondrial DNA for point mutations and deletions in blood and muscle were negative. Sequencing of RRM2B, a gene encoding cytosolic p53-inducible ribonucleoside reductase small subunit (RIR2B), revealed 2 pathogenic mutations, c.329G>A (p.R110H) and c.362G>A (p.R121H). These mutations are predicted to affect the docking interface of the RIR2B homodimer and likely result in impaired enzyme activity.
This study expands the clinical spectrum of impaired RIR2B function, challenges the notion of locus homogeneity of MNGIE, and sheds light on the pathogenesis of conditions involved in the homeostasis of the mitochondrial nucleotide pool. Our findings suggest that patients with MNGIE who have normal thymidine levels should be tested for RRM2B mutations.
Archives of neurology 09/2009; 66(8):1028-32. · 6.31 Impact Factor
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ABSTRACT: Dense liquid phases, metastable with respect to a solid phase, but stable with respect to the solution, have been known to form in solutions of proteins and small-molecule substances. Here, with the protein lumazine synthase as a test system, using dynamic and static light scattering and atomic force microscopy, we demonstrate submicron size clusters of dense liquid. In contrast to the macroscopic dense liquid, these clusters are metastable not only with respect to the crystals, but also with respect to the low-concentration solution: the characteristic cluster lifetime is limited to approximately 10 s, after which they decay. The cluster population is detectable only if they occupy >10(-6) of the solution volume and have a number density >105 cm-3 for 3 to 11% of the monitored time. The cluster volume fraction varies within wide limits and reaches up to 10(-3). Increasing protein concentration increases the frequency of cluster detection but does not affect the ranges of the cluster sizes, suggesting that a preferred cluster size exists. A simple Monte Carlo model with protein-like potentials reproduces the metastable clusters of dense liquid with limited lifetimes and variable sizes and suggests that the mean cluster size is determined by the kinetics of growth and decay and not by thermodynamics.
The Journal of Physical Chemistry B 03/2007; 111(12):3106-14. · 3.70 Impact Factor
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ABSTRACT: The solvent around protein molecules in solutions is structured and this structuring introduces a repulsion in the intermolecular interaction potential at intermediate separations. We use Monte Carlo simulations with isotropic, pair-additive systems interacting with such potentials. We test if the liquid-liquid and liquid-solid phase lines in model protein solutions can be predicted from universal curves and a pair of experimentally determined parameters, as done for atomic and colloid materials using several laws of corresponding states. As predictors, we test three properties at the critical point for liquid-liquid separation: temperature, as in the original van der Waals law, the second virial coefficient, and a modified second virial coefficient, all paired with the critical volume fraction. We find that the van der Waals law is best obeyed and appears more general than its original formulation: A single universal curve describes all tested nonconformal isotropic pair-additive systems. Published experimental data for the liquid-liquid equilibrium for several proteins at various conditions follow a single van der Waals curve. For the solid-liquid equilibrium, we find that no single system property serves as its predictor. We go beyond corresponding-states correlations and put forth semiempirical laws, which allow prediction of the critical temperature and volume fraction solely based on the range of attraction of the intermolecular interaction potential.
The Journal of Physical Chemistry B 10/2006; 110(35):17638-44. · 3.70 Impact Factor
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ABSTRACT: Recent experiments have revealed several surprising features of the phase equilibria in protein solutions: liquid-liquid phase separation which is, in some cases, metastable with respect to the liquid-solid equilibrium, and in others-unobservable; widely varying crystallization enthalpies, including completely athermal crystallization; the co-existence of several crystalline polymorphs; and others. Other studies have shown that the solvent molecules at the hydrophobic and polar patches on the protein molecular surfaces are structured, introducing repulsive forces at surface separations equal to several water molecule sizes. In search of a causal link between the latter and former findings, we apply Monte Carlo simulation techniques in the investigation of phase diagrams associated with globular biological molecules in solution. We account for the solvent structuring via short-range isotropic two-body intermolecular potentials exhibiting multiple extrema. We show that the introduction of a repulsive maximum or a secondary attractive minimum at separations longer than the primary attractive minimum has dramatic effects on the phase diagram: liquid-liquid separation curves are driven to lower or higher temperatures, the sensitivity of the solubility curve (liquidus) to temperature, i.e., the enthalpy of crystallization, is significantly reduced or enhanced, metastable liquid-liquid separation may become stable and vice versa, and both low- and high-density crystalline phases are observed. The similarity of these features of the simulated phase behavior to those observed experimentally suggests that at least some of the mysteries of the protein phase equilibria may be due to the structuring of the solvent around the protein molecular surfaces. Another conclusion is that at least some of the dense liquids seen in protein solutions may be stable and not metastable with respect to a solid phase.
Physical Review E 07/2006; 73(6 Pt 1):061917. · 2.26 Impact Factor
