Structural insights into abasic site for Fpg specific binding and catalysis: comparative high-resolution crystallographic studies of Fpg bound to various models of abasic site analogues-containing DNA

Fpg is a DNA glycosylase that recognizes and excises the mutagenic 8-oxoguanine (8-oxoG) and the potentially lethal formamidopyrimidic residues (Fapy). Fpg is also associated with an AP lyase activity which successively cleaves the abasic (AP) site at the 3′ and 5′ sides by βδ-elimination. Here, we present the high-resolution crystal structures of the wild-type and the P1G defective mutant of Fpg from Lactococcus lactis bound to 14mer DNA duplexes containing either a tetrahydrofuran (THF) or 1,3-propanediol (Pr) AP site analogues. Structures show that THF is less extrahelical than Pr and its backbone C5′–C4′–C3′ diverges significantly from those of Pr, rAP, 8-oxodG and FapydG. Clearly, the heterocyclic oxygen of THF is pushed back by the carboxylate of the strictly conserved E2 residue. We can propose that the ring-opened form of the damaged deoxyribose is the structure active form of the sugar for Fpg catalysis process. Both structural and functional data suggest that the first step of catalysis mediated by Fpg involves the expulsion of the O4′ leaving group facilitated by general acid catalysis (involving E2), rather than the immediate cleavage of the N-glycosic bond of the damaged nucleoside

Study of Convective Heat Transfer in Grinding Applied to Tool Carbide

The grinding temperature is of great importance for the quality and integrity of machined cemented carbide tool. Tool edge surfaces may be damaged by softening or being stressed, hardened, burned, or cracked. Former research on grinding temperature prediction often made assumptions to simplify heat convection due to the grinding fluid. However, these simplifying assumptions can sometimes undermine the mathematical relationships between grinding conditions and surface temperature, particularly in low-temperature grinding where fluid convection is most important. This paper is an attempt to provide an improved comprehensive thermal model for the prediction of contact temperatures and for monitoring and control of thermal damage. Based on previous thermal model research, this paper tackles a key element of the thermal model for temperature prediction. It proposes a convective heat transfer model based on the classic theory of turbulent flow passing a plate. Theoretical predictions from the thermal model of turbulent flow developed in this paper are compared with experimental values. Predictions are further compared with values from a previously published laminar flow model. And it is shown that the new model leads to a significant reduction in predicted temperatures. The results suggest that the thermal model for the turbulent flow provides a reasonable estimate of predicted temperature values within the region of the fluid boiling temperature. The estimates appear to be an improvement compared with the laminar flow thermal model. The turbulent flow thermal model is considered to improve estimates of background contact temperatures in grinding cemented carbide

Using complementary techniques to distinguish cryptic species: A new Erysimum (Brassicaceae) species from North Africa

• Premise of the study : Cryptic species are superfi cially morphologically indistinguishable and therefore erroneously classifi ed under one single name. The identifi cation and delimitation of these species is usually a diffi cult task. The main aim of this study is to provide an inclusive methodology that combines standard and new tools to allow accurate identifi cation of cryptic species. We used Erysimum nervosum s.l. as a model system. • Methods : Four populations belonging to E. nervosum s.l. were sampled at their two distribution ranges in Morocco (the Atlas Mountains and the Rif Mountains). Fifteen individuals per population were collected to assess standard taxonomic traits. Additionally, corolla color and shape were quantifi ed in 30 individuals per population using spectrophotometry and geometric morphometrics, respectively. Finally, we collected tissue samples from each population per species to study the phylogenetic relationships among them. • Key results : Using the standard taxonomic traits, we could not distinguish the four populations. Nonetheless, there were differences in corolla color and shape between plants from the two mountain ranges. The population differentiation based on quantitative morphological differences were confi rmed and supported by the phylogenetic relationships obtained for these populations and the rest of the Moroccan Erysimum species. • Conclusions : The joint use of the results obtained from standard taxonomic traits, quantitative analyses of plant phenotype, and molecular data suggests the occurrence of two species within E. nervosum s.l. in Morocco, one located in the Atlas Mountains ( E. nervosum s.s.) and the other in the Rif Mountains ( E. riphaeanum sp. nov.). Consequently, we suggest that combining quantitative and molecular approaches with standard taxonomy greatly benefi ts the identifi cation of cryptic species

DNA containing a chemically reduced apurinic site is a high affinity ligand for the E.coli formamidopyrimidine-DNA glycosyiase

International audienceThe E. coli Formamidopyrimidine-DNA Glycosylase (FPG protein), a monomeric DNA repair enzyme of 30.2 kDa, was purified to homogeneity in large quantities. The FPG protein excises imidazole ring-opened purines and 8-hydroxyguanine residues from DNA. Besides DNA glycosylase activity, the FPG protein is endowed with an EDTA-resistant activity which nicks DNA at apurinic/apyrimidic sites (AP sites). In contrast, DNAs containing chemically reduced AP sites are not incised by the FPG protein. However, the DNA glycosylase activity of the FPG protein is strongly inhibited in the presence of a purified synthetic 24 base-pair double-stranded oligonucleotide which contains a single apurinic site transformed chemically through borohydride reduction into a ring-opened deoxyribose derivative. The ability of the FPG protein to form a complex with this synthetically modified DNA was studied by electrophoresis in non-denaturing polyacrylamide gels. The FPG protein specifically binds the double-stranded oligonucleotide containing an apurinic site previously reduced in the presence of sodium borohydride. The complex was identified as a single retardation band on non-denaturing polyacrylamide gel electrophoresis. Complex formation is reversible and an apparent dissociation constant, KDapp, of 2.6 x 10(-10) M was determined. In contrast, no such retardation band was obtained between the FPG protein and double-stranded DNA containing an intact apurinic site or single-stranded DNA containing either an intact or a reduced apurinic site

Description of ordered solvent molecules in a platinated decanucleotide duplex refined at 1.6 Å resolution against experimental MAD phases

International audienceAccurate experimental phases derived from a MAD experiment may be useful to enable the identification of solvent molecules during the course of an atomic parameter refinement. The structure of a double-stranded DNA decanucleotide bearing a cisplatin interstrand cross-link at 1.6A resolution, whose phases were first determined experimentally using the L(III) edge of the Pt atom, was refined by various methods. The previously published structure resulted from a least-squares refinement using the structure-factor magnitudes and stereochemical restraints (program SHELX). In this paper, these previous results are compared with a model obtained by the likelihood-maximization method (program REFMAC) which allows the combination of the observed magnitudes with experimental MAD phases. This solution corresponded to a lower R(free) (18.8 compared with 20.3%), a lower R factor and accounted for 135 water molecules and one spermine molecule collected by the program wARP during refinement. The previously published SHELX solution exhibited no spermine molecule and accounted for 92 water molecules, only 74 of which are also present in the model obtained with the MAD phases. In order to verify that these improvements were actually related to the use of the MAD phases, the same type of procedure without the MAD phases was applied starting from the initial model. The resulting solution had a higher R(free) (20.3%), which could be related to the loss of 22 water molecules and the addition of 20 new ones. MAD phases therefore seem especially helpful in preventing the model bias which may affect the solvent molecules. All models have in common a hydration cage of nine water molecules which surround the platinum residue. In addition to the spermine molecule, the model obtained with the MAD phases allows description of the water-molecule organization, with reproducible motifs related to the base pairs and to the phosphodiester backbone