Lesion-Specific DNA-Binding and Repair Activities of Human O⁶-Alkylguanine DNA Alkyltransferase

Binding experiments with alkyl-transfer-active and -inactive mutants of human O6-alkylguanine DNA alkyltransferase (AGT) show that it forms an O6-methylguanine (6mG)-specific complex on duplex DNA that is distinct from non-specific assemblies previously studied. Specific complexes with duplex DNA have a 2:1 stoichiometry that is formed without accumulation of a 1:1 intermediate. This establishes a role for cooperative interactions in lesion binding. Similar specific complexes could not be detected with single-stranded DNA. The small difference between specific and non-specific binding affinities strongly limits the roles that specific binding can play in the lesion search process. Alkyl-transfer kinetics with a single-stranded substrate indicate that two or more AGT monomers participate in the rate-limiting step, showing for the first time a functional link between cooperative binding and the repair reaction. Alkyl-transfer kinetics with a duplex substrate suggest that two pathways contribute to the formation of the specific 6mG-complex; one at least first order in AGT, we interpret as direct lesion binding. The second, independent of [AGT], is likely to include AGT transfer from distal sites to the lesion in a relatively slow unimolecular step. We propose that transfer between distal and lesion sites is a critical step in the repair process

Quaternary Interactions and Supercoiling Modulate the Cooperative DNA Binding of AGT

Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein–protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ωave. We found that while nearest-neighbor contacts were significant, long-range interactions dominated cooperativity and this pattern held true whether the DNA was single-stranded or duplex. Experiments with single plasmid topoisomers showed that the average cooperativity was sensitive to DNA twist, and was strongest when the DNA was slightly underwound. This suggests that AGT proteins are optimally juxtaposed when the DNA is near its torsionally-relaxed state. Most striking was the decline of binding stoichiometry with linking number. As stoichiometry and affinity differences were not correlated, we interpret this as evidence that supercoiling occludes AGT binding sites. These features suggest that AGT\u27s lesion-search distributes preferentially to sites containing torsionally-relaxed DNA, in vivo

Alternating groups and moduli space lifting Invariants

Main Theorem: Spaces of r-branch point 3-cycle covers, degree n or Galois of degree n!/2 have one (resp. two) component(s) if r=n-1 (resp. r\ge n). Improves Fried-Serre on deciding when sphere covers with odd-order branching lift to unramified Spin covers. We produce Hurwitz-Torelli automorphic functions on Hurwitz spaces, and draw Inverse Galois conclusions. Example: Absolute spaces of 3-cycle covers with +1 (resp. -1) lift invariant carry canonical even (resp. odd) theta functions when r is even (resp. odd). For inner spaces the result is independent of r. Another use appears in, http://www.math.uci.edu/~mfried/paplist-mt/twoorbit.html, "Connectedness of families of sphere covers of A_n-Type." This shows the M(odular) T(ower)s for the prime p=2 lying over Hurwitz spaces first studied by, http://www.math.uci.edu/~mfried/othlist-cov/hurwitzLiu-Oss.pdf, Liu and Osserman have 2-cusps. That is sufficient to establish the Main Conjecture: (*) High tower levels are general-type varieties and have no rational points.For infinitely many of those MTs, the tree of cusps contains a subtree -- a spire -- isomorphic to the tree of cusps on a modular curve tower. This makes plausible a version of Serre's O(pen) I(mage) T(heorem) on such MTs. Establishing these modular curve-like properties opens, to MTs, modular curve-like thinking where modular curves have never gone before. A fuller html description of this paper is at http://www.math.uci.edu/~mfried/paplist-cov/hf-can0611591.html .Comment: To appear in the Israel Journal as of 1/5/09; v4 is corrected from proof sheets, but does include some proof simplification in \S

Cooperative Cluster Formation, DNA Bending and Base-Flipping by O\u3csup\u3e6\u3c/sup\u3e-Alkylguanine-DNA Alkyltransferase

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of ≤11 proteins. Longer clusters, predicted by the McGhee–von Hippel model, were not seen even at high [protein]. Interestingly, torsional stress due to DNA unwinding has the potential to limit cluster size to the observed range. DNA at cluster sites showed bend angles (∼0, ∼30 and ∼60°) that are consistent with models in which each protein induces a bend of ∼30°. Distributions of complexes along the DNA are incompatible with sequence specificity but suggest modest preference for DNA ends. These properties tell us about environments in which AGT may function. Small cooperative clusters and the ability to accommodate a range of DNA bends allow function where DNA topology is constrained, such as near DNA-replication complexes. The low sequence specificity allows efficient and unbiased lesion search across the entire genome

Hendra Virus Fusion Protein Transmembrane Domain Contributes to Pre-Fusion Protein Stability

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726–35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability

Association of urinary uromodulin with kidney function decline and mortality: the health ABC study .

BackgroundUrine uromodulin (uUMOD) is a protein secreted by the kidney tubule. Recent studies have suggested that higher uUMOD may be associated with improved kidney and mortality outcomes.MethodsUsing a case-cohort design, we evaluated the association between baseline uUMOD levels and ≥ 30% estimated glomerular filtration rate (eGFR) decline, incident chronic kidney disease (CKD), rapid kidney function decline, and mortality using standard and modified Cox proportional hazards regression.ResultsThe median value of uUMOD was 25.8 µg/mL, mean age of participants was 74 years, 48% were women, and 39% were black. Persons with higher uUMOD had lower prevalence of diabetes and coronary artery disease (CAD), and had lower systolic blood pressure. Persons with higher uUMOD also had higher eGFR, lower urinary albumin to creatinine ratio (ACR), and lower C-reactive protein (CRP). There was no association of uUMOD with > 30% eGFR decline. In comparison to those in the lowest quartile of uUMOD, those in the highest quartile had a significantly (53%) lower risk of incident CKD (CI 73%, 18%) and a 51% lower risk of rapid kidney function decline (CI 76%, 1%) after multivariable adjustment. Higher uUMOD was associated with lower risk of mortality in demographic adjusted models, but not after multivariable adjustment.ConclusionHigher levels of uUMOD are associated with lower risk of incident CKD and rapid kidney function decline. Additional studies are needed in the general population and in persons with advanced CKD to confirm these findings.


Repair of \u3cem\u3eO\u3c/em\u3e\u3csup\u3e6\u3c/sup\u3e-Methylguanine Adducts in Human Telomeric G-Quadruplex DNA by \u3cem\u3eO\u3c/em\u3e\u3csup\u3e6\u3c/sup\u3e-Alkylguanine-DNA Alkyltransferase

O6-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O6-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O6-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ~ 5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs

Crystallization of hepatocyte nuclear factor 4α (HNF4α) in complex with the HNF1α promoter element

Sample preparation, characterization, crystallization and preliminary X-ray analysis are reported for the HNF4α–DNA binary complex

Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association \u3cem\u3eIn Vitro\u3c/em\u3e

Enveloped viruses require viral fusion proteins to promote fusion of the viral envelope with a target cell membrane. To drive fusion, these proteins undergo large conformational changes that must occur at the right place and at the right time. Understanding the elements which control the stability of the prefusion state and the initiation of conformational changes is key to understanding the function of these important proteins. The construction of mutations in the fusion protein transmembrane domains (TMDs) or the replacement of these domains with lipid anchors has implicated the TMD in the fusion process. However, the structural and molecular details of the role of the TMD in these fusion events remain unclear. Previously, we demonstrated that isolated paramyxovirus fusion protein TMDs associate in a monomer-trimer equilibrium, using sedimentation equilibrium analytical ultracentrifugation. Using a similar approach, the work presented here indicates that trimeric interactions also occur between the fusion protein TMDs of Ebola virus, influenza virus, severe acute respiratory syndrome coronavirus (SARS CoV), and rabies virus. Our results suggest that TM-TM interactions are important in the fusion protein function of diverse viral families

Heterogeneous processes: Laboratory, field, and modeling studies

The efficiencies of chemical families such as ClO(x) and NO(x) for altering the total abundance and distribution of stratospheric ozone are controlled by a partitioning between reactive (active) and nonreactive (reservoir) compounds within each family. Gas phase thermodynamics, photochemistry, and kinetics would dictate, for example, that only about 1 percent of the chlorine resident in the lower stratosphere would be in the form of active Cl or ClO, the remainder existing in the reservoir compounds HCl and ClONO2. The consistency of this picture was recently challenged by the recognition that important chemical transformations take place on polar regions: the Airborne Antarctic Ozone Experiment (AAOE) and the Airborne Arctic Stratospheric Expedition (AASA). Following the discovery of the Antarctic ozone hole, Solomon et al. suggested that the heterogeneous chemical reaction: ClONO2(g)+HCl(s) yields Cl2(g)+HNO3(s) could play a key role in converting chlorine from inactive forms into a species (Cl2) that would rapidly dissociate in sunlight to liberate atomic chlorine and initiate ozone depletion. The symbols (s) and (g) denote solid phase, or adsorbed onto a solid surface, and gas phase, respectively, and represent the approach by which such a reaction is modeled rather than the microscopic details of the reaction. The reaction was expected to be most important at altitudes where PSC's were most prevalent (10 to 25 km), thereby extending the altitude range over which chlorine compounds can efficiently destroy ozone from the 35 to 45 km region (where concentrations of active chlorine are usually highest) to lower altitudes where the ozone concentration is at its peak. This chapter will briefly review the current state of knowledge of heterogeneous processes in the stratosphere, emphasizing those results obtained since the World Meteorological Organization (WMO) conference. Sections are included on laboratory investigations of heterogeneous reactions, the characteristics and climatology of PSC's, stratospheric sulfate aerosols, and evidence of heterogeneous chemical processing