Tom Ellenberger

Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution.

DNA polymerases change their specificity for nucleotide substrates with each catalytic cycle, while achieving error frequencies in the range of 10 −5to 10−6. Here we present a 2.2 Å crystal structure of the replicative DNA polymerase from bacteriophage T7 complexed with a primer–template and a nucleoside triphosphate in the polymerase active site. The structure illustrates how nucleotides are selected in a template-directed manner, and provides a structural basis for a metal-assisted mechanism of phosphoryl transfer by a large group of related polymerases.

Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix domain.

Adipocyte determination- and differentiation-dependent factor 1 (ADD1), a member of the basic helix-loop-helix (bHLH) family of transcription factors, has been associated with both adipocyte differentiation and cholesterol homeostasis (in which case it has been termed SREBP1). Using PCR-amplified binding analysis, we demonstrate that ADD1/SREBP1 has dual DNA sequence specificity, binding to both an E-box motif (ATCACGTGA) and a non-E-box sequence previously shown to be important in cholesterol metabolism, sterol regulatory element 1 (SRE-1; ATCACCCCAC). The ADD1/SREBP1 consensus E-box site is similar to a regulatory sequence designated the carbohydrate response element, defined by its ability to regulate transcription in response to carbohydrate in genes involved in fatty acid and triglyceride metabolism in liver and fat. When expressed in fibroblasts, ADD1/SREBP1 activates transcription through both the carbohydrate response E-box element and SRE-1. Substitution of an atypical tyrosine in the basic region of ADD1/SREBP1 to an arginine found in most bHLH protein causes a restriction to only E-box binding. Conversely, substitution of a tyrosine for the equivalent arginine in another bHLH protein, upstream stimulatory factor, allows this factor to acquire a dual binding specificity similar to that of ADD1/SREBP1. Promoter activation by ADD1/SREBP1 through the carbohydrate response element E box is not sensitive to the tyrosine-to-arginine mutation, while activation through SRE-1 is completely suppressed. These data illustrate that ADD1/SREBP1 has dual DNA sequence specificity controlled by a single amino acid residue; this dual specificity may provide a novel mechanism to coordinate different pathways of lipid metabolism.

An open and closed case for all polymerases

The recently determined structures of HIV-1 reverse transcriptase and Taq DNA polymerase in complex with DNA primer-template and an incoming nucleotide have shown that a large conformational change configures the polymerase active site for nucleotidyl transfer. The structure of reverse transcriptase in the catalytic complex will open the path to the rational design of novel nucleoside analog inhibitors of viral replication.

Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7.

Helicases that unwind DNA at the replication fork are ring-shaped oligomeric enzymes that move along one strand of a DNA duplex and catalyze the displacement of the complementary strand in a reaction that is coupled to nucleotide hydrolysis. The helicase domain of the replicative helicase-primase protein from bacteriophage T7 crystallized as a helical filament that resembles the Escherichia coli RecA protein, an ATP-dependent DNA strand exchange factor. When viewed in projection along the helical axis of the crystals, six protomers of the T7 helicase domain resemble the hexameric rings seen in electron microscopic images of the intact T7 helicase-primase. Nucleotides bind at the interface between pairs of adjacent subunits where an arginine is near the gamma-phosphate of the nucleotide in trans. The bound nucleotide stabilizes the folded conformation of a DNA-binding motif located near the center of the ring. These and other observations suggest how conformational changes are coupled to DNA unwinding activity.

Crystal Structure of a Human Alkylbase-DNA Repair Enzyme Complexed to DNA: Mechanisms for Nucleotide Flipping and Base Excision

DNA N-glycosylases are base excision-repair proteins that locate and cleave damaged bases from DNA as the first step in restoring the genetic blueprint. The human enzyme 3-methyladenine DNA glycosylase removes a diverse group of damaged bases from DNA, including cytotoxic and mutagenic alkylation adducts of purines. We report the crystal structure of human 3-methyladenine DNA glycosylase complexed to a mechanism-based pyrrolidine inhibitor. The enzyme has intercalated into the minor groove of DNA, causing the abasic pyrrolidine nucleotide to flip into the enzyme active site, where a bound water is poised for nucleophilic attack. The structure shows an elegant means of exposing a nucleotide for base excision as well as a network of residues that could catalyze the in-line displacement of a damaged base from the phosphodeoxyribose backbone.

Human DNA ligase I completely encircles and partially unwinds nicked DNA.

The end-joining reaction catalysed by DNA ligases is required by all organisms and serves as the ultimate step of DNA replication, repair and recombination processes. One of three well characterized mammalian DNA ligases, DNA ligase I, joins Okazaki fragments during DNA replication. Here we report the crystal structure of human DNA ligase I (residues 233 to 919) in complex with a nicked, 5′ adenylated DNA intermediate. The structure shows that the enzyme redirects the path of the double helix to expose the nick termini for the strand-joining reaction. It also reveals a unique feature of mammalian ligases: a DNA-binding domain that allows ligase I to encircle its DNA substrate, stabilizes the DNA in a distorted structure, and positions the catalytic core on the nick. Similarities in the toroidal shape and dimensions of DNA ligase I and the proliferating cell nuclear antigen sliding clamp are suggestive of an extensive protein–protein interface that may coordinate the joining of Okazaki fragments.

Structural Basis for the Excision Repair of Alkylation-Damaged DNA

Base-excision DNA repair proteins that target alkylation damage act on a variety of seemingly dissimilar adducts, yet fail to recognize other closely related lesions. The 1.8 A crystal structure of the monofunctional DNA glycosylase AlkA (E. coli 3-methyladenine-DNA glycosylase II) reveals a large hydrophobic cleft unusually rich in aromatic residues. An Asp residue projecting into this cleft is essential for catalysis, and it governs binding specificity for mechanism-based inhibitors. We propose that AlkA recognizes electron-deficient methylated bases through pi-donor/acceptor interactions involving the electron-rich aromatic cleft. Remarkably, AlkA is similar in fold and active site location to the bifunctional glycosylase/lyase endonuclease III, suggesting the two may employ fundamentally related mechanisms for base excision.

DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA.

The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 Å crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1‐azaribose abasic nucleotide out of DNA and induces a 66° bend in the DNA with a marked widening of the minor groove. The position of the 1‐azaribose in the enzyme active site suggests an SN1‐type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA–DNA complex offers the first glimpse of a helix–hairpin–helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner.

Crystal structure of the Escherichia coli Rob transcription factor in complex with DNA.

The Escherichia coli Rob protein is a transcription factor belonging to the AraC/XylS protein family that regulates genes involved in resistance to antibiotics, organic solvents and heavy metals. The genes encoding these proteins are activated by the homologous proteins MarA and SoxS, although the level of activation can vary for the different transcription factors. Here we report a 2.7 Å crystal structure of Rob in complex with the micF promoter that reveals an unusual mode of binding to DNA. The Rob–DNA complex differs from the previously reported structure of MarA bound to the mar promoter, in that only one of Robs dual helix-turn-helix (HTH) motifs engages the major groove of the binding site. Biochemical studies show that sequence specific interactions involving only one of Robs HTH motifs are sufficient for high affinity binding to DNA. The two different modes of DNA binding seen in crystal structures of Rob and MarA also match the distinctive patterns of DNA protection by AraC at several sites within the pBAD promoter. These and other findings suggest that gene activation by AraC/XylS transcription factors might involve two alternative modes of binding to DNA in different promoter contexts.

Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase

Accurate DNA replication involves polymerases with high nucleotide selectivity and proofreading activity. We show here why both fidelity mechanisms fail when normally accurate T7 DNA polymerase bypasses the common oxidative lesion 8‐oxo‐7, 8‐dihydro‐2′‐deoxyguanosine (8oG). The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template. A model of a corresponding structure with dATP predicts steric and electrostatic clashes that would reduce but not eliminate insertion of dA. The structure of a postinsertional complex shows 8oG(syn)·dA (anti) in a Hoogsteen‐like base pair at the 3′ terminus, and polymerase interactions with the minor groove surface of the mismatch that mimic those with undamaged, matched base pairs. This explains why translesion synthesis is permitted without proofreading of an 8oG·dA mismatch, thus providing insight into the high mutagenic potential of 8oG.