Bertrand Castaing

Structural basis for the recognition of the FapydG lesion (2,6-diamino-4-hydroxy-5-formamidopyrimidine) by formamidopyrimidine-DNA glycosylase.

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from damaged DNA. Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA. The structure reveals that Fpg stabilizes the cFapydG nucleoside into an extrahelical conformation inside its substrate binding pocket. In contrast to the recognition of the 8-oxodG lesion, which is bound with the glycosidic bond in a syn conformation, the cFapydG lesion displays in the complex an anti conformation. Furthermore, Fpg establishes interactions with all the functional groups of the FapyG base lesion, which can be classified in two categories: (i) those specifying a purine-derived lesion (here a guanine) involved in the Watson-Crick face recognition of the lesion and probably contributing to an optimal orientation of the pyrimidine ring moiety in the binding pocket and (ii) those specifying the imidazole ring-opened moiety of FapyG and probably participating also in the rotameric selection of the FapydG nucleobase. These interactions involve strictly conserved Fpg residues and structural water molecules mediated interactions. The significant differences between the Fpg recognition modes of 8-oxodG and FapydG provide new insights into the Fpg substrate specificity.

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.

Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium

ABSTRACT The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (PramA). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of PramA, including the −10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with PramA as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (KD [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted PramA of an MDR S. Typhimurium clinical isolate than for the wild-type PramA (KD = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.

Radiation Affects Binding of Fpg Repair Protein to an Abasic Site Containing DNA

Abstract Gillard, N., Begusova, M., Castaing, B. and Spotheim-Maurizot, M. Radiation Affects Binding of Fpg Repair Protein to an Abasic Site Containing DNA. Radiat. Res. 162, 566– 571 (2004). During the base excision repair of certain DNA lesions, the formamidopyrimidine-DNA glycosylase (Fpg) binds specifically to the DNA region containing an abasic (AP) site. Is this step affected by exposure to ionizing radiation? To answer this question, we studied a complex between a DNA duplex containing an analogue of an abasic site (the 1,3-propanediol site, Pr) and a mutated Lactococcus lactis Fpg (P1G-LlFpg) lacking strand cleavage activity. Upon irradiation of the complex, the ratio of bound/free partners decreased. When the partners were irradiated separately, the irradiated DNA still bound the unirradiated protein, whereas irradiated Fpg no longer bound unirradiated DNA. Thus irradiation hinders Fpg-DNA binding because of the damage to the protein. Using our radiolytic attack simulation procedure RADACK (Begusova et al., J. Biomol. Struct. Dyn. 19, 141–157, 2001), we reveal the potential hot spots for damage in the irradiated protein. Most of them are essential for the interaction of Fpg with DNA, which explains the radiation-induced loss of binding ability of Fpg. The doses necessary to destroy the complex are higher than those inactivating Fpg irradiated separately. As confirmed by our calculations, this can be explained by the partial protection of the protein by the bound DNA.

Bacterial Base Excision Repair Enzyme Fpg Recognizes Bulky N7-Substituted-FapydG Lesion via Unproductive Binding Mode

Fpg is a bacterial base excision repair enzyme that removes oxidized purines from DNA. This work shows that Fpg and its eukaryote homolog Ogg1 recognize with high affinity FapydG and bulky N7-benzyl-FapydG (Bz-FapydG). The comparative crystal structure analysis of stable complexes between Fpg and carbocyclic cFapydG or Bz-cFapydG nucleoside-containing DNA provides the molecular basis of the ability of Fpg to bind both lesions with the same affinity and to differently process them. To accommodate the steric hindrance of the benzyl group, Fpg selects the adequate rotamer of the extrahelical Bz-cFapydG formamido group, forcing the bulky group to go outside the binding pocket. Contrary to the binding mode of cFapydG, the particular recognition of Bz-cFapydG leads the BER enzymes to unproductive complexes which would hide the lesion and slow down its repair by the NER machinery.

5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases

DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.

Repair of 8-oxo-7,8-dihydroguanine in prokaryotic and eukaryotic cells: Properties and biological roles of the Fpg and OGG1 DNA N-glycosylases ☆

&NA; Oxidatively damaged DNA results from the attack of sugar and base moieties by reactive oxygen species (ROS), which are formed as byproducts of normal cell metabolism and during exposure to endogenous or exogenous chemical or physical agents. Guanine, having the lowest redox potential, is the DNA base the most susceptible to oxidation, yielding products such as 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) and 2–6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FapyG). In DNA, 8‐oxoG was shown to be mutagenic yielding GC to TA transversions upon incorporation of dAMP opposite this lesion by replicative DNA polymerases. In prokaryotic and eukaryotic cells, 8‐oxoG is primarily repaired by the base excision repair pathway (BER) initiated by a DNA N‐glycosylase, Fpg and OGG1, respectively. In Escherichia coli, Fpg cooperates with MutY and MutT to prevent 8‐oxoG‐induced mutations, the “GO‐repair system”. In Saccharomyces cerevisiae, OGG1 cooperates with nucleotide excision repair (NER), mismatch repair (MMR), post‐replication repair (PRR) and DNA polymerase &eegr; to prevent mutagenesis. Human and mouse cells mobilize all these pathways using OGG1, MUTYH (MutY‐homolog also known as MYH), MTH1 (MutT‐homolog also known as NUDT1), NER, MMR, NEILs and DNA polymerases &eegr; and &lgr;, to prevent 8‐oxoG‐induced mutations. In fact, mice deficient in both OGG1 and MUTYH develop cancer in different organs at adult age, which points to the critical impact of 8‐oxoG repair on genetic stability in mammals. In this review, we will focus on Fpg and OGG1 proteins, their biochemical and structural properties as well as their biological roles. Other DNA N‐glycosylases able to release 8‐oxoG from damaged DNA in various organisms will be discussed. Finally, we will report on the role of OGG1 in human disease and the possible use of 8‐oxoG DNA N‐glycosylases as therapeutic targets. Graphical abstract Figure. No caption available. Highlights8‐oxo‐7,8‐dihydroguanine (8‐oxoG) in DNA is a major cause of genetic instability.Fpg and OGG1 DNA N‐glycosylases initiate 8‐oxoG‐repair in prokaryotes and eukaryotes.Structural, catalytic and biological properties of Fpg and OGG1 proteins.8‐oxoG‐repair in eukaryotes is mediated by a complex enzymatic network.Inactivation of 8‐oxoG‐repair results in cancer predisposition in mice and human.

Insights into the DNA repair process by the formamidopyrimidine-DNA glycosylase investigated by molecular dynamics.

Formamidopyrimidine‐DNA glycosylase (Fpg) identifies and removes 8‐oxoguanine from DNA. All of the X‐ray structures of Fpg complexed to an abasic site containing DNA exhibit a common disordered region present in the C‐terminal domain of the enzyme. However, this region is believed to be involved in the damaged base binding site when the initial protein/DNA complex is formed. The dynamic behavior of the disordered polypeptide (named Loop) in relation to the supposed scenario for the DNA repair mechanism was investigated by molecular dynamics on different models, derived from the X‐ray structure of Lactococcus lactis Fpg bound to an abasic site analog‐containing DNA and of Bacillus stearothermophilus Fpg bound to 8‐oxoG. This study shows that the presence of the damaged base influences the dynamics of the whole enzyme and that the Loop location is dependent on the presence and on the conformation of the 8‐oxoG in its binding site. In addition, from our results, the conformation of the 8‐oxoG seems to be favored in syn in the L. lactis models, in agreement with the available X‐ray structure from B. stearothermophilus Fpg and with a possible catalytic role of the flexibility of the Loop region.

The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNA

Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere.

Complex oligomeric structure of a truncated form of DdrA: A protein required for the extreme radiotolerance of Deinococcus

In order to preserve their genome integrity, organisms have developed elaborate tactics for genome protection and repair. The Deinococcus radiodurans bacteria famous for their extraordinary tolerance toward high doses of radiations or long period of desiccation, possess some specific genes with unknown function which are related to their survival in such extreme conditions. Among them, ddrA is an orphan gene specific of Deinococcus genomes. DdrA, the product of this gene was suggested to be a component of the DNA end protection system. Here we provide a three-dimensional reconstruction of the Deinococcus deserti DdrA((1-160)) by electron microscopy. Although not functional in vivo, this truncated protein keeps its DNA binding ability at the wild-type level. DdrA((1-160)) has a complex three-dimensional structure based on a heptameric ring that can self-associate to form a larger molecular weight assembly. We suggest that the complex architecture of DdrA plays a role in the substrate specificity and favors an efficient DNA repair.