Biomedical subjects
Gregory L Verdine
Publications and source records attributed to Gregory L Verdine.
DNA lesion recognition by the bacterial repair enzyme MutM.
MutM is a bacterial DNA glycosylase that removes the mutagenic lesion 8-oxoguanine (oxoG) from duplex DNA. The means of oxoG recognition by MutM (also known as Fpg) is of fundamental interest, in light of the vast excess of normal guanine bases present in genomic DNA. The crystal structure of a recognition-competent but catalytically inactive version of MutM in complex with oxoG-containing DNA reveals the structural basis for recognition. MutM binds the oxoG nucleoside in the syn glycosidic configuration and distinguishes oxoG from guanine by reading out the protonation state of the N7 atom. The segment of MutM principally responsible for oxoG recognition is a flexible loop, suggesting that conformational mobility influences lesion recognition and catalysis. Furthermore, the structure of MutM in complex with DNA containing an alternative substrate, dihydrouracil, demonstrates how MutM is able to recognize lesions other than oxoG.
Structure of a trapped endonuclease III-DNA covalent intermediate.
Nearly all cells express proteins that confer resistance to the mutagenic effects of oxidative DNA damage. The primary defense against the toxicity of oxidative nucleobase lesions in DNA is the base-excision repair (BER) pathway. Endonuclease III (EndoIII) is a [4Fe-4S] cluster-containing DNA glycosylase with repair activity specific for oxidized pyrimidine lesions in duplex DNA. We have determined the crystal structure of a trapped intermediate that represents EndoIII frozen in the act of repairing DNA. The structure of the protein-DNA complex provides insight into the ability of EndoIII to recognize and repair a diverse array of oxidatively damaged bases. This structure also suggests a rationale for the frequent occurrence in certain human cancers of a specific mutation in the related DNA repair protein MYH.
Extensively stereodiversified scaffolds for use in diversity-oriented library synthesis.
The syntheses of stereodiverse libraries of 12 and 19 are reported, where each asterisk represents an independently varied stereocenter. These scaffolds provide additional templates for investigations of geometric diversity in library syntheses. Libraries of these N-Fmoc-amino acids were further functionalized by incorporation into a peptide sequence, demonstrating the utility of 12 and 19 as building blocks for diversity oriented synthesis.
Unpredictable stereochemical preferences for mu opioid receptor activity in an exhaustively stereodiversified library of 1,4-enediols.
Using olefin cross-metathesis, we synthesized a novel stereodiversified library of compounds 3 containing a trans-1,4-enediol. Screening this library for mu opioid receptor (MOR) affinity identified multiple high-affinity ligands and revealed that the stereochemical configuration varied widely among those ligands having the highest affinity. It was not possible to predict the configurations of the most active compounds 3 on the basis of the configuration of endomorphin-2, a known MOR peptide ligand, validating the diversity-based approach to ligand discovery.
2,6-Dimethyltyrosine analogues of a stereodiversified ligand library: highly potent, selective, non-peptidic mu opioid receptor agonists.
We recently reported the use of an exhaustively stereodiversified library based on endomorphin-2 (1) to discover mu opioid receptor (MOR) ligands of type 2-4. Here, we report the synthesis and evaluation of 2,6-dimethyltyrosine analogues 5-10. These analogues showed improved affinity for MOR relative to 2-4. In the cases of 5 and 6, we synthesized and evaluated five stereoisomers of each, thereby discovering stereoisomers with unexpected potency, selectivity, and efficacy. These results illustrate the utility of acyclic, stereodiverse libraries.
Structural and biochemical exploration of a critical amino acid in human 8-oxoguanine glycosylase.
Members of the HhH-GPD superfamily of DNA glycosylases are responsible for the recognition and removal of damaged nucleobases from DNA. The hallmark of these proteins is a motif comprising a helix-hairpin-helix followed by a Gly/Pro-rich loop and terminating in an invariant, catalytically essential aspartic acid residue. In this study, we have probed the role of this Asp in human 8-oxoguanine DNA glycosylase (hOgg1) by mutating it to Asn (D268N), Glu (D268E), and Gln (D268Q). We show that this aspartate plays a dual role, acting both as an N-terminal alpha-helix cap and as a critical residue for catalysis of both base excision and DNA strand cleavage by hOgg1. Mutation of this residue to asparagine, another helix-capping residue, preserves stability of the protein while drastically reducing enzymatic activity. A crystal structure of this mutant is the first to reveal the active site nucleophile Lys249 in the presence of lesion-containing DNA; this structure offers a tantalizing suggestion that base excision may occur by cleavage of the glycosidic bond and then attachment of Lys249. Mutation of the aspartic acid to glutamine and glutamic acid destabilizes the protein fold to a significant extent but, surprisingly, preserves catalytic activity. Crystal structures of these mutants complexed with an unreactive abasic site in DNA reveal these residues to adopt a sterically disfavored helix-capping conformation.
Converting the sacrificial DNA repair protein N-ada into a catalytic methyl phosphotriester repair enzyme.
Mutation of the active-site residue Cys38 of N-Ada converts it from a sacrificial DNA repair protein to an enzyme that uses methanethiol as an external sacrificial reagent to repair DNA methyl phosphotriesters catalytically.
Structure and specificity of the vertebrate anti-mutator uracil-DNA glycosylase SMUG1.
Cytosine deamination is a major promutagenic process, generating G:U mismatches that can cause transition mutations if not repaired. Uracil is also introduced into DNA via nonmutagenic incorporation of dUTP during replication. In bacteria, uracil is excised by uracil-DNA glycosylases (UDG) related to E. coli UNG, and UNG homologs are found in mammals and viruses. Ung knockout mice display no increase in mutation frequency due to a second UDG activity, SMUG1, which is specialized for antimutational uracil excision in mammalian cells. Remarkably, SMUG1 also excises the oxidation-damage product 5-hydroxymethyluracil (HmU), but like UNG is inactive against thymine (5-methyluracil), a chemical substructure of HmU. We have solved the crystal structure of SMUG1 complexed with DNA and base-excision products. This structure indicates a more invasive interaction with dsDNA than observed with other UDGs and reveals an elegant water displacement/replacement mechanism that allows SMUG1 to exclude thymine from its active site while accepting HmU.
Product-assisted catalysis in base-excision DNA repair.
Most spontaneous damage to bases in DNA is corrected through the action of the base-excision DNA repair pathway. Base excision repair is initiated by DNA glycosylases, lesion-specific enzymes that intercept aberrant bases in DNA and catalyze their excision. How such proteins accomplish the feat of catalyzing no fewer than five sequential reaction steps using a single active site has been unknown. To help answer this, we report the structure of a trapped catalytic intermediate in DNA repair by human 8-oxoguanine DNA glycosylase. This structure and supporting biochemical results reveal that the enzyme sequesters the excised lesion base and exploits it as a cofactor to participate in catalysis. To our knowledge, the present example represents the first documented case of product-assisted catalysis in an enzyme-catalyzed reaction.
Covalent trapping of protein-DNA complexes.
High-resolution structural studies of protein-DNA complexes have proven to be an invaluable means of understanding the diverse functions of proteins that manage the genome. Most of the structures determined to date represent proteins bound noncovalently to various DNA sequences or structures. Although noncovalent complexation is often adequate to study the structures of proteins that have robust, specific interactions with DNA, it is poorly suited to the study of transient intermediates in enzyme-catalyzed DNA processing reactions or of complexes that exist in multiple equilibrating forms. In recent years, strategies developed for the covalent trapping of protein-DNA complexes have begun to show promise as a window into an otherwise inaccessible world of structure.
High-affinity mu opioid receptor ligands discovered by the screening of an exhaustively stereodiversified library of 1,5-enediols.
In this communication, we report the synthesis of an exhaustively stereodiversified library of 16 1,5-enediols (2) and the screening of these compounds for mu opioid receptor (MOR) binding. The stereochemical configuration of 2 strongly impacted the binding affinity, and (S,S,S,R)-2 exhibited a Ki of 8.8 nM for MOR, comparable to that of endomorphin-2 (Ki = 1.2 nM). Moreover, compounds 2 exhibited 5-86-fold selectivity for MOR over delta opioid receptor (DOR) and 16-150-fold selectivity for MOR over kappa opioid receptor (KOR). Additionally, analogues of 2 were synthesized which showed the importance of the trans olefin for receptor binding but that modifications of the C-terminal amino acid were well tolerated. Ligand 11 is noteworthy because it retains only one of the amide bonds present in 1, but binds MOR with an affinity of 10 nM and 110- and 600-fold selectivity for MOR over DOR and KOR. These results demonstrate the utility of stereochemical diversity in the discovery of bioactive small molecules.
5-amino-2'-deoxyuridine, a novel thymidine analogue for high-resolution footprinting of protein-DNA complexes.
[formula: see text] 5-Amino-2'-deoxyuridine 5'-triphosphate, an analogue of deoxythymidine triphosphate, was synthesized and found to be a substrate of Taq DNA polymerase. The DNA-borne analogue underwent selective chemical reaction with permanganate. The use of 5-amino-dU as an interference probe was validated using the Ada protein/ada promoter complex. The performance of 5-amino-dU in interference footprints is similar to that of the previously described analogue 5-hydroxy-dU, but the former is incorporated more readily into DNA during enzymatic polymerization.
A genotyping strategy based on incorporation and cleavage of chemically modified nucleotides.
Aiming to facilitate the analysis of human genetic variations in the context of disease susceptibility and varied drug response, we have developed a genotyping method that entails incorporation of a chemically labile nucleotide by PCR followed by specific chemical cleavage of the resulting amplicon at the modified bases. The identity of the cleaved fragments determines the genotype of the DNA. This method, termed Incorporation and Complete Chemical Cleavage, utilizes modified nucleotides 7-deaza-7-nitro-dATP, 7-deaza-7-nitro-dGTP, 5-hydroxy-dCTP, and 5-hydroxy-dUTP, which have increased chemical reactivity but are able to form standard Watson-Crick base pairs. Thus one analog is substituted for the corresponding nucleotide during PCR, generating amplicons that contain nucleotide analogs at each occurrence of the selected base throughout the target DNA except for the primer sequences. Subsequent treatment with an oxidant followed by an organic base results in chemical cleavage at each site of modification, which produces fragments of different lengths and/or molecular weights that reflect the genotype of the original DNA sample at the site of interest. This incorporation and cleavage chemistry are widely applicable to many existing nucleic acid analysis platforms, including gel electrophoresis and mass spectrometry. By combining DNA amplification and analog incorporation into one step, this strategy eliminates preamplification, DNA-strand separation, primer extension, and purification procedures associated with traditional chain-termination chemistry and therefore presents significant advantages in terms of speed, cost, and simplicity of genotyping.
Direct visualization of a DNA glycosylase searching for damage.
DNA glycosylases preserve the integrity of genetic information by recognizing damaged bases in the genome and catalyzing their excision. It is unknown how DNA glycosylases locate covalently modified bases hidden in the DNA helix amongst vast numbers of normal bases. Here we employ atomic-force microscopy (AFM) with carbon nanotube probes to image search intermediates of human 8-oxoguanine DNA glycosylase (hOGG1) scanning DNA. We show that hOGG1 interrogates DNA at undamaged sites by inducing drastic kinks. The sharp DNA bending angle of these non-lesion-specific search intermediates closely matches that observed in the specific complex of 8-oxoguanine-containing DNA bound to hOGG1. These findings indicate that hOGG1 actively distorts DNA while searching for damaged bases.
Trapping distinct structural states of a protein/DNA interaction through disulfide crosslinking.
The N-terminal domain of the Escherichia coli Ada protein (N-Ada) repairs methyl phosphotriesters in DNA through a zinc-mediated transfer to Cys38 of the protein. Methylation of Cys38 enhances the sequence-specific DNA affinity of N-Ada by approximately 1000-fold, thereby enabling the protein to activate the genes of a methylation-resistance regulon. It is of interest to understand the structural basis for metalloactivated methyl transfer and methylation-dependent enhancement of DNA binding activity. Although recent progress has been made on the structural front, efforts to develop a complete picture of N-Ada structure/function have been hampered by the inability to prepare homogeneous protein/DNA complexes representing different states of the unmethylated protein. Here, we describe the development of an approach to trap both sequence-specific and nonsequence-specific DNA recognition complexes of N-Ada through formation of an intermolecular disulfide crosslink between the protein and DNA.
High-resolution footprinting of sequence-specific protein-DNA contacts.
Gene transcription is regulated by proteins that bind specific DNA sequences and control the initiation of RNA synthesis. A major challenge is to map all of the regulatory sites in the genome and to identify the proteins that bind them. Because members of transcription factor families often exhibit similar sequence preferences, methods for determining intermolecular contacts in protein-DNA interfaces must be sensitive to even subtle structural differences. The most detailed structural views of protein-DNA interfaces have been obtained through X-ray crystallography and NMR spectroscopy, and these methods have revolutionized the understanding of the structural determinants of sequence-specific recognition. Neither crystallography nor NMR, however, is particularly well-suited to high-throughput applications such as pan-genomic elucidation of regulatory sequences; in addition, these methods yield no information on the energetic contribution of particular contacts. Here we report a straightforward, high-resolution biochemical method for mapping, at single-nucleotide resolution, DNA bases that are subject to sequence-specific contacts by regulatory proteins.
Structural insights into lesion recognition and repair by the bacterial 8-oxoguanine DNA glycosylase MutM.
MutM is a bacterial 8-oxoguanine glycosylase responsible for initiating base-excision repair of oxidized guanine residues in DNA. Here we report five different crystal structures of MutM-DNA complexes that represent different steps of the repair reaction cascade catalyzed by the protein and also differ in the identity of the base opposite the lesion (the 'estranged' base). These structures reveal that the MutM active site performs the multiple steps of base-excision and 3' and 5' nicking with minimal rearrangement of the DNA backbone.