Carlos de los Santos

Michigan Molecular Interactions (MiMI): putting the jigsaw puzzle together

Protein interaction data exists in a number of repositories. Each repository has its own data format, molecule identifier and supplementary information. Michigan Molecular Interactions (MiMI) assists scientists searching through this overwhelming amount of protein interaction data. MiMI gathers data from well-known protein interaction databases and deep-merges the information. Utilizing an identity function, molecules that may have different identifiers but represent the same real-world object are merged. Thus, MiMI allows the users to retrieve information from many different databases at once, highlighting complementary and contradictory information. To help scientists judge the usefulness of a piece of data, MiMI tracks the provenance of all data. Finally, a simple yet powerful user interface aids users in their queries, and frees them from the onerous task of knowing the data format or learning a query language. MiMI allows scientists to query all data, whether corroborative or contradictory, and specify which sources to utilize. MiMI is part of the National Center for Integrative Biomedical Informatics (NCIBI) and is publicly available at: .

NMR Characterization of a DNA duplex containing the major acrolein-derived deoxyguanosine adduct gamma-OH-1,N2-propano-2'-deoxyguanosine

The environmental and endogenous mutagen acrolein reacts with cellular DNA to produce several isomeric 1,N 2-propanodeoxyguanosine adducts. High resolution NMR spectroscopy was used to establish the structural features of the major acrolein-derived adduct, γ-OH-1,N 2-propano-2′-deoxyguanosine. In aqueous solution, this adduct was shown to assume a ring-closed form. In contrast, when γ-OH-1,N 2-propano-2′-deoxyguanosine pairs with dC at the center of an 11-mer oligodeoxynucleotide duplex, the exocyclic ring opens, enabling the modified base to participate in a standard Watson-Crick base pairing alignment. Analysis of the duplex spectra reveals a regular right-handed helical structure with all residues adopting an anti orientation around the glycosidic torsion angle and Watson-Crick alignments for all base pairs. We conclude from this study that formation of duplex DNA triggers the hydrolytic conversion of γ-OH-1,N 2-propano-2′-deoxyguanosine to an open chain form, a structure that facilitates pairing with dC during DNA replication and accounts for the surprising lack of mutagenicity associated with this DNA adduct.

An Improved Reaction Coordinate for Nucleic Acid Base Flipping Studies.

Base flipping is a common strategy utilized by many enzymes to gain access to the functional groups of nucleic acid bases in duplex DNA which are otherwise protected by the DNA backbone and hydrogen bonding with their partner bases. Several X-ray crystallography studies have revealed flipped conformations of nucleotides bound to enzymes. However, little is known about the base-flipping process itself, even less about the role of the enzymes. Computational studies have used umbrella sampling to elicit the free energy profile of the base-flipping process using a pseudodihedral angle to represent the reaction coordinate. In this study, we have used an unrestrained trajectory in which a flipped base spontaneously reinserted into the helix in order to evaluate and improve the previously defined pseudodihedral angle. Our modified pseudodihedral angles use a new atom selection to improve the numerical stability of the restraints and also provide better correlation to the extent of flipping observed in simulations. Furthermore, on the basis of the comparison of potential of mean force (PMF) generated using different reaction coordinates, we observed that the shape of a flipping PMF profile is strongly dependent on the definition of the reaction coordinate, even for the same data set.

Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition

Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination.

Structure and stability of DNA containing an aristolactam II-dA lesion: implications for the NER recognition of bulky adducts

Aristolochic acids I and II are prevalent plant toxicants found in the Aristolochiaceae plant family. Metabolic activation of the aristolochic acids leads to the formation of a cyclic N-hydroxylactam product that can react with the peripheral amino group of purine bases generating bulky DNA adducts. These lesions are mutagenic and established human carcinogens. Interestingly, although AL-dG adducts progressively disappear from the DNA of laboratory animals, AL-dA lesions has lasting persistence in the genome. We describe here NMR structural studies of an undecameric duplex damaged at its center by the presence of an ALII-dA adduct. Our data establish a locally perturbed double helical structure that accommodates the bulky adduct by displacing the counter residue into the major groove and stacking the ALII moiety between flanking bases. The presence of the ALII-dA perturbs the conformation of the 5′-side flanking base pair, but all other pairs of the duplex adopt standard conformations. Thermodynamic studies reveal that the lesion slightly decreases the energy of duplex formation in a sequence-dependent manner. We discuss our results in terms of its implications for the repair of ALII-dA adducts in mammalian cells.

Energetic Preference of 8-oxoG Eversion Pathways in a DNA Glycosylase

Base eversion is a fundamental process in the biochemistry of nucleic acids, allowing proteins engaged in DNA repair and epigenetic modifications to access target bases in DNA. Crystal structures reveal end points of these processes, but not the pathways involved in the dynamic process of base recognition. To elucidate the pathway taken by 8-oxoguanine during base excision repair by Fpg, we calculated free energy surfaces during eversion of the damaged base through the major and minor grooves. The minor groove pathway and free energy barrier (6-7 kcal/mol) are consistent with previously reported results (Qi, Y.; Spong, M. C.; Nam, K.; Banerjee, A.; Jiralerspong, S.; Karplus, M.; Verdine, G. L. Nature 2009, 462, 762.) However, eversion of 8-oxoG through the major groove encounters a significantly lower barrier (3-4 kcal/mol) more consistent with experimentally determined rates of enzymatic sliding during lesion search (Blainey, P. C.; van Oijent, A. M.; Banerjee, A.; Verdine, G. L.; Xie, X. S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 5752.). Major groove eversion has been suggested for other glycosylases, suggesting that in addition to function, dynamics of base eversion may also be conserved.

Effect of 6-thioguanine on the stability of duplex DNA

The incorporation of 6-thioguanine (S6G) into DNA is a prerequisite for its cytotoxic action, but duplex structure is not significantly perturbed by the presence of the lesion [J. Bohon and C. R. de los Santos (2003) Nucleic Acids Res., 31, 1331–1338]. It is therefore possible that the mechanism of cytotoxicity relies on a loss of stability rather than a pathway involving direct structural recognition. The research described here focuses on the changes in thermodynamic properties of duplex DNA owing to the introduction of S6G as well as the kinetic properties of base pairs involving S6G. Replacement of a guanine in a G•C pair by S6G results in ∼1 kcal/mol less favorable Gibbs free energy of duplex formation at 37°C. S6G•T and G•T mismatch-containing duplexes have almost identical Gibbs free energy at 37°C, with values ∼3 kcal/mol less favorable than that of the control. Base pair stability is affected by S6G. The lifetime of the normal G•C base pair is ∼125 ms, whereas that of the G•T mismatch is below the detection limit. The lifetimes of S6G•C and S6G•T pairs are ∼7 and 2 ms, respectively, demonstrating that, although S6G significantly decreases the stability of the pairing with cytosine, it slightly increases that of a mismatch.

Incorporation of 3-aminobenzanthrone into 2'-deoxyoligonucleotides and its impact on duplex stability.

3-Nitrobenzanthrone (3NBA), an environmental pollutant and potent mutagen, causes DNA damage via the reaction of its metabolically activated form with the exocyclic amino groups of purines and the C-8 position of guanine. The present work describes a synthetic approach to the preparation of oligomeric 2′-deoxyribonucleotides containing a 2-(2′-deoxyguanosin-N2-yl)-3-aminobenzanthrone moiety, one of the major DNA adducts found in tissues of living organisms exposed to 3NBA. The NMR spectra indicate that the damaged oligodeoxyribonucleotide is capable of forming a regular double helical structure with the polyaromatic moiety assuming a single conformation at room temperature; the spectra suggest that the 3ABA moiety resides in the duplex minor groove pointing toward the 5′-end of the modified strand. Thermodynamic studies show that the dG(N2)-3ABA lesion has a stabilizing effect on the damaged duplex, a fact that correlates well with the long persistence of this damage in living organisms.

Molecular mechanics parameters for the FapydG DNA lesion

FapydG is a common oxidative DNA lesion involving opening of the imidazole ring. It shares the same precursor as 8‐oxodG and can be excised by the same enzymes as 8‐oxodG. However, the loss of the aromatic imidazole in FapydG results in a reduction of the double bond character between C5 and N7, with an accompanying increase in conformational flexibility. Experimental characterization of FapydG is hampered by high reactivity, and thus it is desirable to investigate structural details through computer simulation. We show that the existing Amber force field parameters for FapydG do not reproduce X‐ray structural data. We employed quantum mechanics energy profile calculations to derive new molecular mechanics parameters for the rotation of the dihedral angles in the eximidazole moiety. Using these parameters, all‐atom simulations in explicit water reproduce the nonplanar conformation of cFapydG in the crystal structure of the complex with L. lactis glycosylase Fpg. We note that the nonplanar structure is stabilized by an acidic residue that is not present in most Fpg sequences. Simulations of the E→S mutant, as present in E. coli, resulted in a more planar conformation, suggesting that the highly nonplanar form observed in the crystal structure may not have direct biological relevance for FapydG.

Solution structure of DNA containing α-OH-PdG: the mutagenic adduct produced by acrolein

Acrolein is a cell metabolic product and a main component of cigarette smoke. Its reaction with DNA produces two guanine lesions γ-OH-PdG, a major adduct that is nonmutagenic in mammalian cells, and the positional isomer α-OH-PdG. We describe here the solution structure of a short DNA duplex containing a single α-OH-PdG lesion, as determined by solution NMR spectroscopy and restrained molecular dynamics simulations. The spectroscopic data show a mostly regular right-handed helix, locally perturbed at its center by the presence of the lesion. All undamaged residues of the duplex are in anti orientation, forming standard Watson–Crick base-pair alignments. Duplication of proton signals near the damaged site differentiates two enantiomeric duplexes, thus establishing the exocyclic nature of the lesion. At the lesion site, α-OH-PdG rotates to a syn conformation, pairing to its counter cytosine residue that is protonated at pH 5.9. Three-dimensional models produced by restrained molecular dynamics simulations show different hydrogen-bonding patterns between the lesion and its cytosine partner and identify further stabilization of α-OH-PdG in a syn conformation by intra-residue hydrogen bonds. We compare the α-OH-PdG•dC duplex structure with that of duplexes containing the analogous lesion propano-dG and discuss the implications of our findings for the mutagenic bypass of acrolein lesions.