Steven D. Bruner

Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA.

Spontaneous oxidation of guanine residues in DNA generates 8-oxoguanine (oxoG). By mispairing with adenine during replication, oxoG gives rise to a G·C → T·A transversion, a frequent somatic mutation in human cancers. The dedicated repair pathway for oxoG centres on 8-oxoguanine DNA glycosylase (hOGG1), an enzyme that recognizes oxoG·C base pairs, catalysing expulsion of the oxoG and cleavage of the DNA backbone. Here we report the X-ray structure of the catalytic core of hOGG1 bound to oxoG·C-containing DNA at 2.1 Å resolution. The structure reveals the mechanistic basis for the recognition and catalytic excision of DNA damage by hOGG1 and by other members of the enzyme superfamily to which it belongs. The structure also provides a rationale for the biochemical effects of inactivating mutations and polymorphisms in hOGG1. One known mutation, R154H, converts hOGG1 to a pro-mutator by relaxing the specificity of the enzyme for the base opposite oxoG.

Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily

BACKGROUND Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins. RESULTS Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G. CONCLUSIONS S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.

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.

Type II Thioesterase Restores Activity of a NRPS Module Stalled with an Aminoacyl-S-enzyme that Cannot Be Elongated

Nonribosomal peptide synthetases (NRPSs) carry out the biosynthesis of numerous peptide natural products, including many with important clinical applications. The NRPS, organized into a series of modules, is an efficient, high-fidelity assembly line for the production of a particular peptide. Each module consists of domains, whose activities contribute to the accuracy of these assembly-line systems. The activation (A) domain uses ATP to selectively load an amino acid onto the module through formation of a thioester bond to the pantetheine arm of the thiolation (T) domain. Peptide-bond formation, catalyzed by the condensation (C) domain, is stringent for both sidechain identity and stereochemistry. The C domain accepts an aminoacylor peptidylthioester from the preceding module for nucleophilic addition by the amine of the loaded amino acid; this generates the elongated peptide attached to the downstream module. The peptide product is synthesized one amino acid at a time until it reaches the final module. There, the fully synthesized chain is released by a type I thioesterase (TEI), the terminal domain of the NRPS assembly. Despite the high fidelity of this process, an error in any step of the assembly-line synthesis severely impacts the efficiency of the system and creates a bottleneck that results in a buildup of unprocessed intermediates. For example, an error by the A domain, which can load amino acids other than that normally accepted by the C domain, would prevent peptide-bond formation. The loaded module would be blocked until the incorrect amino acid was hydrolyzed (Figure 1). A type II thioesterase (TEII), whose gene is associated with the gene cluster of many NRPSs and related polyketide synthases (PKSs), improves the efficiency of product formation in these systems and has been proposed to edit modules through hydrolysis of acyl groups. In the surfactin NRPS, TEII was shown to regenerate misacylated modules resulting from priming of the apomodule with acyl-CoA groups. In this study we provide evidence to expand the editing function of TEIIs to include restoring the activity of modules stalled by loaded amino acids that cannot be processed. N-acetylcysteamine (SNAC) thioesters have been used previously to assay NRPS domain activities. 13–15] Hydrolysis of SNAC substrates was used here to explore the specificity of the TEII from the tyrocidine biosynthetic operon, TycF. TycF accepted a broad variety of aminoacyl-SNACs of different sidechain identity and stereochemistry with a 20-fold kcat/Km range between the mostand least-active substrate (Table 1). A series of peptidyl-SNACs derived from the tyrocidine sequence was

How do DNA repair proteins locate damaged bases in the genome

The genome is susceptible to the attack of reactive species that chemically modify the bases of DNA. If genetic information is to be transmitted faithfully to successive generations, it is essential that the genome be repaired. All organisms express proteins specifically dedicated to this task. But how do these proteins find the aberrant bases amongst the enormous number of normal ones?

Repair of oxidatively damaged guanine in Saccharomyces cerevisiae by an alternative pathway

BACKGROUND Transversion mutations are caused by 8-oxoguanine (OG), a DNA lesion produced by the spontaneous oxidation of guanine nucleotides, which mis-pairs with adenine during replication. Resistance to this mutagenic threat is mediated by the GO system, the components of which are functionally conserved in bacteria and mammals. To date, only one of three GO system components has been identified in the budding yeast Saccharomyces cerevisiae, namely the OG:C-specific glycosylase/lyase yOgg1. Furthermore, S. cerevisiae has been reported to contain a unique glycosylase/lyase activity, yOgg2, which excises OG residues opposite adenines. Paradoxically, according to the currently accepted model, yOgg2 activity should increase the mutagenicity of OG lesions. Here we report the isolation of yOgg2 and the elucidation of its role in oxidative mutagenesis. RESULTS Borohydride-dependent cross-linking using an OG-containing oligonucleotide substrate led to the isolation of yOgg1 and a second protein, Ntg1, which had previously been shown to process oxidized pyrimidines in DNA. We demonstrate that Ntg1 has OG-specific glycosylase/lyase activity indistinguishable from that of yOgg2. Targeted disruption of the NTG1 gene resulted in complete loss of yOgg2 activity and yeast lacking NTG1 had an elevated rate of A:T to C:G transversions. CONCLUSIONS The Ntg1 and yOgg2 activities are encoded by a single gene. We propose that yOgg2 has evolved to process OG:A mis-pairs that have arisen through mis-incorporation of 8-oxo-dGTP during replication. Thus, the GO system in S. cerevisiae is fundamentally distinct from that in bacteria and mammals.

Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis

Enzyme-catalysed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.

Structural Basis for Phosphopantetheinyl Carrier Domain Interactions in the Terminal Module of Nonribosomal Peptide Synthetases

Phosphopantetheine-modified carrier domains play a central role in the template-directed, biosynthesis of several classes of primary and secondary metabolites. Fatty acids, polyketides, and nonribosomal peptides are constructed on multidomain enzyme assemblies using phosphopantetheinyl thioester-linked carrier domains to traffic and activate building blocks. The carrier domain is a dynamic component of the process, shuttling pathway intermediates to sequential enzyme active sites. Here, we report an approach to structurally fix carrier domain/enzyme constructs suitable for X-ray crystallographic analysis. The structure of a two-domain construct of Escherichia coli EntF was determined with a conjugated phosphopantetheinyl-based inhibitor. The didomain structure is locked in an active orientation relevant to the chemistry of nonribosomal peptide biosynthesis. This structure provides details into the interaction of phosphopantetheine arm with the carrier domain and the active site of the thioesterase domain.

Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophile Thermobifida fusca

Bacteria belonging to the order Actinomycetales have proven to be an important source of biologically active and often therapeutically useful natural products. The characterization of orphan biosynthetic gene clusters is an emerging and valuable approach to the discovery of novel small molecules. Analysis of the recently sequenced genome of the thermophilic actinomycete Thermobifida fusca revealed an orphan nonribosomal peptide biosynthetic gene cluster coding for an unknown siderophore natural product. T. fusca is a model organism for the study of thermostable cellulases and is a major degrader of plant cell walls. Here, we report the isolation and structure elucidation of the fuscachelins, siderophore natural products produced by T. fusca. In addition, we report the purification and biochemical characterization of the termination module of the nonribosomal peptide synthetase. Biochemical analysis of adenylation domain specificity supports the assignment of this gene cluster as the producer of the fuscachelin siderophores. The proposed nonribosomal peptide biosynthetic pathway exhibits several atypical features, including a macrocyclizing thioesterase that produces a 10-membered cyclic depsipeptide and a nonlinear assembly line, resulting in the unique heterodimeric architecture of the siderophore natural product.

Structure and noncanonical chemistry of nonribosomal peptide biosynthetic machinery

Structural biology has provided significant insights into the complex chemistry and macromolecular organization of nonribosomal peptide synthetases. In addition, novel pathways are continually described, expanding the knowledge of known biosynthetic chemistry.