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T Ellenberger

Publications and source records attributed to T Ellenberger.

At least 19 recordsLinked to original sources
PubMed2001

Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7.

The gene 2.5 protein (gp2.5) of bacteriophage T7 is a single-stranded DNA (ssDNA) binding protein that has essential roles in DNA replication and recombination. In addition to binding DNA, gp2.5 physically interacts with T7 DNA polymerase and T7 primase-helicase during replication to coordinate events at the replication fork. We have determined a 1.9-A crystal structure of gp2.5 and show that it has a conserved OB-fold (oligosaccharide/oligonucleotide binding fold) that is well adapted for interactions with ssDNA. Superposition of the OB-folds of gp2.5 and other ssDNA binding proteins reveals a conserved patch of aromatic residues that stack against the bases of ssDNA in the other crystal structures, suggesting that gp2.5 binds to ssDNA in a similar manner. An acidic C-terminal extension of the gp2.5 protein, which is required for dimer formation and for interactions with the T7 DNA polymerase and the primase-helicase, appears to be flexible and may act as a switch that modulates the DNA binding affinity of gp2.5.

Amino Acid Sequence↗
PubMed2001

Tuning DNA "strings": modulating the rate of DNA replication with mechanical tension.

Recent experiments have measured the rate of replication of DNA catalyzed by a single enzyme moving along a stretched template strand. The dependence on tension was interpreted as evidence that T7 and related DNA polymerases convert two (n = 2) or more single-stranded template bases to double helix geometry in the polymerization site during each catalytic cycle. However, we find structural data on the T7 enzyme--template complex indicate n = 1. We also present a model for the "tuning" of replication rate by mechanical tension. This model considers only local interactions in the neighborhood of the enzyme, unlike previous models that use stretching curves for the entire polymer chain. Our results, with n = 1, reconcile force-dependent replication rate studies with structural data on DNA polymerase complexes.

Bacteriophage T7↗
PubMed2001

A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization.

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.

Amino Acid Sequence↗
PubMed2001

Base excision and DNA binding activities of human alkyladenine DNA glycosylase are sensitive to the base paired with a lesion.

The human alkyladenine DNA glycosylase has a broad substrate specificity, excising a structurally diverse group of damaged purines from DNA. To more clearly define the structural and mechanistic bases for substrate specificity of human alkyladenine DNA glycosylase, kinetics of excision and DNA binding activities were measured for several different damaged and undamaged purines within identical DNA sequence contexts. We found that 1,N(6)-ethenoadenine (epsilonA) and hypoxanthine (Hx) were excised relatively efficiently, whereas 7,8-dihydro-8-oxoguanine, O(6)-methylguanine, adenine, and guanine were not. Single-turnover kinetics of excision of Hx and epsilonA paired with T showed that excision of Hx was about four times faster than epsilonA, whereas binding assays showed that the binding affinity was about five times greater for epsilonA than for Hx. The opposing pyrimidine base had a significant effect on the kinetics of excision and DNA binding affinity of Hx but a small effect on those for epsilonA. Surprisingly, replacing a T with a U opposite Hx dramatically reduced the excision rate by a factor of 15 and increased the affinity by a factor of 7-8. The binding affinity of human alkyladenine DNA glycosylase to a DNA product containing an abasic site was similar to that for an Hx lesion.

Adenosine↗
PubMed2001

Crystallizing thoughts about DNA base excision repair.

Chemically damaged bases are removed from DNA by glycosylases that locate the damage and cleave the bond between the modified base and the deoxyribose sugar of the DNA backbone. The detection of damaged bases in DNA poses two problems: (1) The aberrant bases are mostly buried within the double helix, and (2) a wide variety of chemically different modifications must be efficiently recognized and removed. The human alkyladenine glycosylase (AAG) and Escherichia coli Alka DNA glycosylases excise many different types of alkylated bases from DNA. Crystal structures of these enzymes show how substrate bases are exposed to the enzyme active site and they suggest mechanisms of catalytic specificity. Both enzymes bend DNA and flip substrate bases out of the double helix and into the enzyme active site for cleavage. Although AAG and AlkA have very different overall folds, some common features of their substrate-binding sites suggest related strategies for the selective recognition of a chemically diverse group of alkylated substrates.

Alkylation↗
PubMed2001

Crystal structure of a DinB family error-prone DNA polymerase from Sulfolobus solfataricus.

A new group of error-prone DNA polymerases overcomes the blockage posed to normal DNA replication by damaged template bases, suggesting an active site with a loose, flexible pocket that accommodates aberrant DNA structures. We have determined a 2.8 A resolution crystal structure of the Sulfolobus solfataricus Dbh protein, a DNA translesion polymerase closely related to Escherichia coli DNA polymerase IV and human polymerase kappa. A high error rate is observed for the Dbh polymerase in a range of 10(-2)-10(-3) for all 12 base substitution mispairs. The crystal structure of Dbh reveals an overall architecture resembling other DNA polymerases but has unique features that are likely to contribute to error-prone synthesis, including -1 frameshifting mutations.

Amino Acid Sequence↗
PubMed2000

Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG.

The human 3-methyladenine DNA glycosylase [alkyladenine DNA glycosylase (AAG)] catalyzes the first step of base excision repair by cleaving damaged bases from DNA. Unlike other DNA glycosylases that are specific for a particular type of damaged base, AAG excises a chemically diverse selection of substrate bases damaged by alkylation or deamination. The 2.1-A crystal structure of AAG complexed to DNA containing 1,N(6)-ethenoadenine suggests how modified bases can be distinguished from normal DNA bases in the enzyme active site. Mutational analyses of residues contacting the alkylated base in the crystal structures suggest that the shape of the damaged base, its hydrogen-bonding characteristics, and its aromaticity all contribute to the selective recognition of damage by AAG.

Base Pairing↗
PubMed2000

Structural studies of human alkyladenine glycosylase and E. coli 3-methyladenine glycosylase.

Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair glycosylases that recognize and excise a variety of alkylated bases from DNA. The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases. Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond. In contrast to AAG, the structure of the AlkA-DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a S(N)1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.

Alkylation↗
PubMed2000

Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides.

We have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7. The structure reveals how subunit contacts stabilize the hexamer. Deviation from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a "binding change" mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer. This model suggests a mechanism for DNA translocation.

Bacteriophage T7↗
PubMed2000

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 A 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 degrees 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 S(N)1-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.

Amino Acid Motifs↗
PubMed2000

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 A 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 Rob's 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 Rob's 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.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

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.

Binding Sites↗
PubMed1998

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.

Alkylation↗
PubMed1998

Recognition of core-type DNA sites by lambda integrase.

Escherichia coli phage lambda integrase (Int) is a 40 kilodalton, 356 amino acid residue protein, which belongs to the lambda Int family of site-specific recombinases. The amino-terminal domain (residues 1 to 64) of Int binds to "arm-type" DNA sites, distant from the sites of DNA cleavage. The carboxy-terminal fragment, termed C65 (residues 65 to 356), binds "core-type" DNA sites and catalyzes cleavage and ligation at these sites. It has been further divided into two smaller domains, encompassing residues 65 to 169 and 170 to 356, respectively. The latter has been characterized and its crystal structure has been determined. Although this domain catalyzes the cleavage and rejoining of DNA strands it, unexpectedly, does not form electrophorectically stable complexes with core-type DNA. Here we have investigated the critical features of lambda Int binding to core-type DNA sites; especially, the role of the central 65 to 169 domain. To eliminate the complexities arising from lambda Int's heterobivalency we studied Int C65, which was shown to be as competent as Int, in binding to, and cleaving, core-type sites. Zero-length UV crosslinking was used to show that Ala125 and Ala126 make close contact with bases in the core-type DNA. Modification by pyridoxal 5'-phosphate was used to identify Lys103 at the protein-DNA interface. Since both of the identified loci are in the central domain, it was cloned and purified and found to bind to core-type DNA autonomously and specifically. The synergistic roles of the catalytic and the central, or core-binding (CB), domains in the interaction with core-type DNA are discussed for (Int and related DNA recombinases.

Bacteriophage lambda↗
PubMed1998

DNA-mediated folding and assembly of MyoD-E47 heterodimers.

Basic region helix-loop-helix (bHLH) transcription factors regulate key steps in early development by binding to regulatory DNA sites as heterodimers consisting of a tissue-specific factor and a widely expressed factor. We have examined the folding, dimerization, and DNA binding properties of the muscle-specific bHLH protein MyoD and its partner E47, to understand why these proteins preferentially associate in heterodimeric complexes with DNA. In the absence of DNA, the E47 bHLH domain forms a very stable homodimer, whereas MyoD is unfolded and monomeric. Fluorescence quenching experiments show that MyoD does not dimerize with E47 under dilute conditions in the absence of DNA. Residues in and around the loop of the E47 bHLH domain contribute to its markedly greater stability. An altered MyoD bHLH substituted with the loop segment from E47 folds in the absence of DNA, and it readily dimerizes with E47. In the presence of a specific DNA binding site, MyoD and E47 both form homodimeric complexes with DNA that have similar dissociation constants, despite the very different stabilities of these protein dimers off DNA. A 1:1 mixture of these bHLH domains forms almost exclusively heterodimeric complexes on DNA. Assembly of these bHLH-DNA complexes is apparently governed by the strength of each subunit's interaction with the DNA and not by the strength of protein-protein interactions at the dimer interface. These findings suggest that preferential association of MyoD with E47 in DNA complexes results from more favorable DNA contacts made by one or both subunits of the heterodimer in comparison with either homodimeric complex.

Amino Acid Sequence↗