Dominique Burnouf

kinITC: A New Method for Obtaining Joint Thermodynamic and Kinetic Data by Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is the method of choice for obtaining thermodynamic data on a great variety of systems. Here we show that modern ITC apparatus and new processing methods allow researchers to obtain a complete kinetic description of systems more diverse than previously thought, ranging from simple ligand binding to complex RNA folding. We illustrate these new features with a simple case (HIV-1 reverse transcriptase/inhibitor interaction) and with the more complex case of the folding of a riboswitch triggered by the binding of its ligand. The originality of the new kinITC method lies in its ability to dissect, both thermodynamically and kinetically, the two components: primary ligand binding and subsequent RNA folding. We are not aware of another single method that can yield, in a simple way, such deep insight into a composite process. Our study also rationalizes common observations from daily ITC use.

Observing Translesion Synthesis of an Aromatic Amine DNA Adduct by a High-fidelity DNA Polymerase

Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2′-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2′-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.

Distinct β‐clamp interactions govern the activities of the Y family PolIV DNA polymerase

The prototypic Y family DNA polymerase IV (PolIV) of Escherichia coli is involved in multiple replication‐associated processes including spontaneous mutagenesis, translesion synthesis (TLS), cell fitness, survival under stressful conditions and checkpoint like functions. It interacts physically and functionally with the replisomes β processivity clamp through the canonical PolIV C‐terminal peptide (CTP). A second interaction that involves a portion of the little finger (LF) domain of PolIV has been structurally described. Here we show that the LF–β interaction stabilizes the clamp–polymerase complex in vitro and is necessary for the access of PolIV to ongoing replication forks in vivo. However, in contrast to the CTP–β, the LF–β interaction is dispensable for the role of the polymerase in TLS. This discloses two independent modes of action for PolIV and, in turn, uncovers a novel way by which the cell may regulate the potentially deleterious effect of such low fidelity polymerases during replication.

Mismatch repair genes and microsatellite instability as molecular markers for gynecological cancer detection

Due to major developments in genetics over the past decade, molecular biology tests are serving promising tools in early diagnosis and follow-up of cancer patients. Recent epidemiological studies revealed that the risk for each individual to develop cancer is closely linked to his/her own genetic potentialities. Some populations that are defective in DNA repair processes, for example in Xeroderma pigmentosum or in the Lynch syndrome, are particularly prone to cancer due to the accumulation of mutations within the genome. Such populations would benefit from the development of tests aimed at identifying people who are particularly at risk. Here, we review some data suggesting that the inactivation of mismatch repair is often found in endometrial cancer and we discuss molecular-based strategies that would help to identify the affected individuals in families with cases of glandular malignancies.

Thermodynamics of HIV-1 reverse transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors.

HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a reinterpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.

Differential Modes of Peptide Binding onto Replicative Sliding Clamps from Various Bacterial Origins.

Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interact efficiently with our designed peptides and assemble the Escherichia coli and related orthologs clamps, whereas group II clamps poorly interact with the same peptides and include Bacillus subtilis and other Gram-positive clamps. These studies also suggest that the peptide binding process could occur via different mechanisms, which depend on the type of clamp.

Structure-Based Design of Short Peptide Ligands Binding onto the E. coli Processivity Ring

The multimeric DNA sliding clamps confer high processivity to replicative DNA polymerases and are also binding platforms for various enzymes involved in DNA metabolism. These enzymes interact with the clamp through a small peptide that binds into a hydrophobic pocket which is a potential target for the development of new antibacterial compounds. Starting from a generic heptapeptide, we used a structure-based strategy to improve the design of new peptide ligands. Chemical modifications at specific residues result in a dramatic increase of the interaction as measured by SPR and ITC. The affinity of our best hits was improved by 2 orders of magnitude as compared to the natural ligand, reaching 10(-8) M range. The molecular basis of the interactions was analyzed by solving the co-crystal structures of the most relevant peptides bound to the clamp and reveals how chemical modifications establish new contacts and contributes to an increased affinity of the ligand.

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches.

ABSTRACT Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

A single N-2-acetylaminofluorene adduct alters the footprint of T7 (exo−) DNA polymerase bound to a model primer–template junction

Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footprint T7 (exo-) DNA polymerase bound to a model primer-template junction. The polymerase was blocked at a specific position either by the omission of dCTP from the reaction mix or by the presence of a N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has been shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replication of AAF-monomodified single-stranded vectors. The footprints obtained with unmodified primer-template DNA define two protected domains separated by an inter-region that remains sensitive to DNaseI, and several hypersensitive sites located on both strands. Binding of the polymerase to AAF monomodified duplexes results in the same protection pattern as that obtained with the unmodified duplexes. However, the hypersensitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the duplex DNA within the polymerase cleft.

Molecular Aspects of Repair and Mutagenesis of cis-Platinum-Induced Lesions

The conversion of DNA lesions into mutations is an active biochemical process. Due to the remarkable efficiency of the error free repair mechanisms only a very small number of DNA lesions will eventually be processed into a mutation. Indeed, under normal conditions, less than one percent of DNA lesions give rise to mutations. This makes the biochemical study of the mechanisms involved in mutagenesis very difficult. As a first molecular approach, the study of the mutational specificity of a given mutagen will provide important informations concerning the mechanisms that are involved. This is particularly true if the analysis of the mutational specificity can be performed in hosts having altered genotypes for repair and (or) mutagenesis. Bacteria are in this respect the organisms of choice, due to the large number of existing repair, replication and recombination mutants.