No abstract

No abstract

SUMMARY Numerous studies of the anti-HIV-1 envelope glycoprotein 41 (gp41) broadly neutralizing antibody 4E10 suggest that 4E10 also interacts with membrane lipids, but the antibody regions contacting lipids and its orientation with respect to the viral membrane are unknown. Vaccine immunogens capable of re-eliciting these membrane proximal external region (MPER)-like antibodies may require a lipid component to be successful. We performed a systematic crystallographic study of lipid binding to 4E10 to identify lipids bound by the antibody and the lipid-interacting regions. We identified phosphatidic acid, phosphatidylglycerol, and glycerol phosphate as specific ligands for 4E10 in the crystal structures. 4E10 used its CDRH1 loop to bind the lipid head groups, while its CDRH3 interacted with the hydrophobic lipid tails. Identification of the lipid binding sites on 4E10 may aid design of immunogens for vaccines that include a lipid component in addition to the MPER on gp41 for generation of broadly neutralizing antibodies.

Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design.

We examined the effect of a single O 6 -methylguanine (O 6 -MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O 6 -MeG, with C being the major product (ϳ70%) and T or A being the minor species (ϳ20% or ϳ10%, respectively), consistent with steady-state kinetic parameters. Transient-state kinetic experiments revealed that k pol , the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O 6 -MeG was ϳ6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre-steady state. The lack of any structural information regarding how O 6 -MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that "wobble" pairing occurs between C and O 6 -MeG. A structure containing T opposite O 6 -MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher K m,dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O 6 -MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site.Of the myriad forms that covalent modification of DNA can take, alkylation of the purine/pyrimidine bases is one of the most extensively studied (1, 2). The term "alkylating agent" encompasses a variety of known carcinogenic chemicals ranging from the spontaneously reactive nitrogen and sulfur mustards (e.g. mechlorethamine) and N-alkyl-N-nitrosoureas to metabolically activated compounds such as cyclophosphamide and N-nitrosamines (3). Guanine is generally considered the most easily oxidized of the bases, and the N7 position is the most nucleophilic atom of guanine. One prevalent form of guanine oxidation occurs at the O6 position, with a simple and widely studied process being addition of a methyl group to form O 6 -MeG 2 (4). Methylation of the O6 atom results in alternate pairing schemes that include a O 6 -MeG:C "wobble" pairing and a pseudo-"Watson-Crick" O 6 -MeG:T pair ( Fig. 1), and the relevance of O 6 -MeG to mutagenesis is well established (5-7). Cells can repair O 6 -MeG by recognition and/or removal of the lesion through either the mismatch repair pathway or through the actions of (O 6 -alkylguanine DNA alkyltransferase) (8 -10). O 6 -MeG is even observed in the DNA of the general population, although the level measured between studies has varied (4, 11). Substantially increased levels of O 6 -MeG are found in patients treated with chemotherapeutic regimes that include methylating agents (4, 12, 13).Of the enzymes associated with what has commonly been referred to as "translesion synthesis," the Y-family DNA polymerases are thought to represent the major constituent present during post-replication repair of covalently modified DNA (14 -16). Four human Y-family polymerases ar...

Human polymerase kappa (hPol ) is one of four eukaryotic Y-class DNA polymerases and may be an important element in the cellular response to polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which can lead to reactive oxygenated metabolite-mediated oxidative stress. Here, we present a detailed analysis of the activity and specificity of hPol bypass opposite the major oxidative adduct 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxoG). Unlike its archaeal homolog Dpo4, hPol bypasses this lesion in an error-prone fashion by inserting mainly dATP. Analysis of transient-state kinetics shows diminished "bursts" for dATP:8-oxoG and dCTP:8-oxoG incorporation, indicative of non-productive complex formation, but dATP:8-oxoG insertion events that do occur are 2-fold more efficient than dCTP:G insertion events. Crystal structures of ternary hPol complexes with adducted template-primer DNA reveal non-productive (dGTP and dATP) alignments of incoming nucleotide and 8-oxoG. Structural limitations placed upon the hPol by interactions between the N-clasp and finger domains combined with stabilization of the syn-oriented template 8-oxoG through the side chain of Met-135 both appear to contribute to error-prone bypass. Mutating Leu-508 in the little finger domain of hPol to lysine modulates the insertion opposite 8-oxoG toward more accurate bypass, similar to previous findings with Dpo4. Our structural and activity data provide insight into important mechanistic aspects of error-prone bypass of 8-oxoG by hPol compared with accurate and efficient bypass of the lesion by Dpo4 and polymerase .DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery, and various mechanisms exist to either remove the resulting lesions or bypass them in a more or less mutation-prone fashion (1). Error-prone polymerases are central to trans-lesion synthesis across sites of damaged DNA (2, 3). Four so-called Y-class DNA polymerases have been identified in humans, Pol , 4 Pol , Pol , and Rev1, which exhibit different activities and abilities to replicate past a flurry of individual lesions (4, 5). Homologs have also been identified and characterized in other organisms, notably DinB (Pol IV) in Escherichia coli (6 -8), Dbh in Sulfolobus acidocaldarius (9, 10), and Dpo4 in Sulfolobus solfataricus (11,12). A decade of investigations directed at the structural and functional properties of bypass polymerases have significantly improved our understanding of this class of enzymes (5, 13). A unique feature of Y-class polymerases, compared with the common right-handed arrangement of palm, thumb, and finger subdomains of high fidelity (i.e. A-class) DNA polymerases (14), is a "little finger" or "PAD" (palm-associated domain) subdomain that plays a crucial role in lesion bypass (12,(15)(16)(17)(18)(19)(20)(21). In addition to the little finger subdomain at the C-terminal end of the catalytic core, both Rev1 and Pol exhibit an N-terminal extension that is absent in other translesion polym...

International audienceWe have studied the nonlinear absorption spectra and optical limitingproperties of 2,2′-(9,9-dihexyl) bifluorene. Measurements were done in chloroform solution, in the visible range (450–650 nm), and for nanosecond time-duration pulses. The two-photon absorption(TPA)spectrum, obtained by the up-conversion fluorescence method, shows a resonance at λ=534 nm with a cross-section σ TPA =60 10 −50 cm 4 ⋅s /photon-molecule. Semiempirical quantum chemistry calculation on fluorene and bifluorene suggests an enhancement of the bifluorene TPA due to coupling effects between monomers. In nonlinear transmission measurements,two-photon absorption is reinforced by the excited-state absorption that occurs during the pulse duration. At resonance, the three-photon absorption coefficient is α 3 =14 000 cm 3 /GW 2 for a bifluorene concentration of 600 g/L. This strong nonlinear absorption leads to an efficient optical power limiting in the green and blue parts of the spectrum. The maximum transmitted energy is lower than 10 μJ for an input energy of up to 200 μJ in a F/5 optical geometry

No abstract