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Tom Ellenberger

Publications and source records attributed to Tom Ellenberger.

At least 37 records · Page 2Linked to original sources
PubMed2004

Multiple interactions with the Rad51 recombinase govern the homologous recombination function of Rad54.

In eukaryotes, Rad51 and Rad54 functionally cooperate to mediate homologous recombination and the repair of damaged chromosomes by recombination. Rad51, the eukaryotic counterpart of the bacterial RecA recombinase, forms filaments on single-stranded DNA that are capable of pairing the bound DNA with a homologous double-stranded donor to yield joint molecules. Rad54 enhances the homologous DNA pairing reaction, and this stimulatory effect involves a physical interaction with Rad51. Correspondingly, the ability of Rad54 to hydrolyze ATP and introduce superhelical tension into covalently closed circular plasmid DNA is stimulated by Rad51. By controlled proteolysis, we show that the amino-terminal region of yeast Rad54 is rather unstructured. Truncation mutations that delete the N-terminal 113 or 129 amino acid residues of Rad54 attenuate or ablate physical and functional interactions with Rad51 under physiological ionic strength, respectively. Surprisingly, under less stringent conditions, the Rad54 Delta129 protein can interact with Rad51 in affinity pull-down and functional assays. These results highlight the functional importance of the N-terminal Rad51 interaction domain of Rad54 and reveal that Rad54 contacts Rad51 through separable epitopes.

Adenosine Triphosphate↗
PubMed2004

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.

Bacteriophage T7↗
PubMed2004

Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7.

Ultraviolet-induced DNA damage poses a lethal block to replication. To understand the structural basis for this, we determined crystal structures of a replicative DNA polymerase from bacteriophage T7 in complex with nucleotide substrates and a DNA template containing a cis-syn cyclobutane pyrimidine dimer (CPD). When the 3' thymine is the templating base, the CPD is rotated out of the polymerase active site and the fingers subdomain adopts an open orientation. When the 5' thymine is the templating base, the CPD lies within the polymerase active site where it base-pairs with the incoming nucleotide and the 3' base of the primer, while the fingers are in a closed conformation. These structures reveal the basis for the strong block of DNA replication that is caused by this photolesion.

Bacteriophage T7↗
PubMed2004

A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis.

The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.

Alanine↗
PubMed2004

The Escherichia coli 3-methyladenine DNA glycosylase AlkA has a remarkably versatile active site.

3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision. In contrast to the highly specific 3-methyladenine DNA glycosylase I (E. coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O(2) adducts of thymidine and cytidine. We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro. Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine. These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate. Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs. Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site. The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA.

Base Pair Mismatch↗
PubMed2004

Two structural features of lambda integrase that are critical for DNA cleavage by multimers but not by monomers.

Despite many years of genetic and biochemical studies on the lambda integrase (Int) recombination system, it is still not known whether the Int protein is competent for DNA cleavage as a monomer. We have addressed this question, as part of a larger study of Int functions critical for the formation of higher-order complexes, by isolating "multimer-specific" mutants. We identify a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates. Allele-specific suppressor mutations were generated at these residues. Their interaction with wild-type Int on preformed Holliday junctions indicates that the mutated residues comprise an intermolecular salt bridge. We have also shown that the most C-terminal seven residues of Int, which comprise another previously identified subunit interface, inhibit DNA cleavage by monomeric but not multimeric Int. Taken together, our results lead us to conclude that Int can cleave DNA as a monomer. We also identify and discuss unique structural features of Int that act negatively to reduce its activity as a monomer and other features that act positively to enhance its activity as a multimer.

Amino Acid Substitution↗
PubMed2003

Dissecting the broad substrate specificity of human 3-methyladenine-DNA glycosylase.

Human alkyladenine-DNA glycosylase (AAG) catalyzes the excision of a broad range of modified bases, protecting the genome from many types of alkylative and oxidative DNA damage. We have investigated how AAG discriminates against normal DNA bases, while accommodating a structurally diverse set of lesioned bases, by measuring the rates of AAG-catalyzed (k(st)) and spontaneous N-glycosidic bond hydrolysis (k(non)) for damaged and undamaged DNA oligonucleotides. The rate enhancements for excision of different bases reveal that AAG is most adept at excising the deaminated lesion hypoxanthine (k(st)/k(non) = 10(8)), suggesting that enzymatic activity may have evolved in response to this lesion. Comparisons of the rate enhancements for excision of normal and modified purine nucleobases provide evidence that AAG excludes the normal purines via steric clashes with the exocyclic amino groups of adenine and guanine. However, methylated purines are more chemically labile, and only modest rate enhancements are required for their efficient excision. Base flipping also contributes to specificity as destabilized mismatched base pairs are better substrates than stable Watson-Crick pairs, and many of the lesions recognized by AAG are compromised in their ability to base pair. These findings suggest that AAG reconciles a broad substrate tolerance with the biological imperative to avoid normal DNA by excluding normal bases from the active site rather than by specifically recognizing each lesion.

Adenine↗
PubMed2003

Human alkyladenine DNA glycosylase uses acid-base catalysis for selective excision of damaged purines.

Human alkyladenine DNA glycosylase (AAG) protects against alkylative and oxidative DNA damage, flipping damaged nucleotides out of double-stranded DNA and catalyzing the hydrolytic cleavage of the N-glycosidic bond to release the damaged nucleobase. The crystal structure of AAG bound to a DNA substrate reveals features of the active site that could discriminate between oxidatively damaged or alkylated purines and normal purines. A water molecule bound in the active site adjacent to the anomeric carbon of the N-glycosidic bond is suggestive of direct attack by water, with Glu125 acting as a general base. However, biochemical evidence for this proposed mechanism has been lacking. The structure also fails to explain why smaller pyrimidine nucleosides are excluded as substrates from this relatively permissive active site that catalyzes the excision of a structurally diverse group of damaged purine bases. We have used pH dependencies, site-directed mutagenesis, and a variety of substrates to investigate the catalytic mechanism of AAG. Single-turnover excision of hypoxanthine and 1,N(6)-ethenoadenine follows bell-shaped pH-rate profiles, indicating that AAG-catalyzed excision of these neutral lesions requires the action of both a general acid and a general base. In contrast, the pH-rate profile for the reaction of 7-methylguanine, a positively charged substrate, shows only a single ionization corresponding to a general base. These results suggest that AAG activates neutral lesions by protonation of the nucleobase leaving group. Glu125 must be deprotonated in the active form of the enzyme, consistent with a role as a general base that activates and positions a water nucleophile. Acid-base catalysis can account for much of the 10(8)-fold rate enhancement that is achieved by AAG in the excision of hypoxanthine. The prominent role of nucleobase protonation in catalysis by AAG provides a rationale for its specialization toward damaged purines while effectively excluding pyrimidines.

Acids↗
PubMed2003

Crystal structures of 3-methyladenine DNA glycosylase MagIII and the recognition of alkylated bases.

DNA glycosylases catalyze the excision of chemically modified bases from DNA. Although most glycosylases are specific to a particular base, the 3-methyladenine (m3A) DNA glycosylases include both highly specific enzymes acting on a single modified base, and enzymes with broader specificity for alkylation-damaged DNA. Our structural understanding of these different enzymatic specificities is currently limited to crystal and NMR structures of the unliganded enzymes and complexes with abasic DNA inhibitors. Presented here are high-resolution crystal structures of the m3A DNA glycosylase from Helicobacter pylori (MagIII) in the unliganded form and bound to alkylated bases 3,9-dimethyladenine and 1,N6-ethenoadenine. These are the first structures of a nucleobase bound in the active site of a m3A glycosylase belonging to the helix-hairpin-helix superfamily. MagIII achieves its specificity for positively-charged m3A not by direct interactions with purine or methyl substituent atoms, but rather by stacking the base between two aromatic side chains in a pocket that excludes 7-methylguanine. We report base excision and DNA binding activities of MagIII active site mutants, together with a structural comparison of the HhH glycosylases.

Amino Acid Sequence↗
PubMed2003

Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder.

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.

Bacteriophage lambda↗
PubMed2003

Structure of the coiled-coil dimerization motif of Sir4 and its interaction with Sir3.

The yeast silent information regulators Sir2, Sir3, and Sir4 physically interact with one another to establish a transcriptionally silent state by forming repressive chromatin structures. The Sir4 protein contains binding sites for both Sir2 and Sir3, and these protein-protein interactions are required for gene silencing. Here, we report the X-ray structure of the coiled-coil dimerization motif within the C-terminus of Sir4 and show that it forms a stable 1:1 complex with a dimeric fragment of Sir3 (residues 464-978). We have identified a cluster of residues on the surface of the Sir4 coiled coil required for specific interactions with Sir3. The histone deacetylase Sir2 can also bind to this complex, forming a ternary complex with the truncated Sir3 and Sir4 proteins. The dual interactions of Sir4 with Sir3 and Sir2 suggest a physical basis for recruiting Sir3 to chromatin by virtue of its interactions with Sir4 and with deacetylated histones in chromatin.

Amino Acid Sequence↗
PubMed2003

Arm sequences contribute to the architecture and catalytic function of a lambda integrase-Holliday junction complex.

lambda integrase (Int) mediates recombination between attachment sites on lambda phage and E. coli DNAs. With the assistance of accessory proteins that induce DNA loops, Int bridges pairs of distinct arm- and core-type DNA binding sites to form synapsed recombination complexes, which then recombine via a Holliday junction (HJ) intermediate. We show that, in addition to promoting the proper positioning of Int protomers, the arm sequences facilitate the catalytic activities of the Int tetramer, independent of accessory proteins or physical continuity between the arm and core sites. We have determined the architecture of ternary complexes containing a HJ, Int, and P'1,2 arm-type DNA. These structures accommodate simultaneous binding of Int to direct-repeat arm sites and indirect-repeat core sites and afford a new view of the higher-order recombinogenic complexes.

Bacteriophage lambda↗
PubMed2003

Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis.

DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg(2+) ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the primase active site and then delivering the primed DNA template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.

Amino Acid Sequence↗
PubMed2003

A conformational switch controls the DNA cleavage activity of lambda integrase.

The bacteriophage lambda integrase protein (lambda Int) belongs to a family of tyrosine recombinases that catalyze DNA rearrangements. We have determined a crystal structure of lambda Int complexed with a cleaved DNA substrate through a covalent phosphotyrosine bond. In comparison to an earlier unliganded structure, we observe a drastic conformational change in DNA-bound lambda Int that brings Tyr342 into the active site for cleavage of the DNA in cis. A flexible linker connects the central and the catalytic domains, allowing the protein to encircle the DNA. Binding specificity is achieved through direct interactions with the DNA and indirect readout of the flexibility of the att site. The conformational switch that activates lambda Int for DNA cleavage exposes the C-terminal 8 residues for interactions with a neighboring Int molecule. The protein interactions mediated by lambda Int's C-terminal tail offer a mechanism for the allosteric control of cleavage activity in higher order lambda Int complexes.

Bacteriophage lambda↗
PubMed2003

The crystal structure of the bifunctional primase-helicase of bacteriophage T7.

Within minutes after infecting Escherichia coli, bacteriophage T7 synthesizes many copies of its genomic DNA. The lynchpin of the T7 replication system is a bifunctional primase-helicase that unwinds duplex DNA at the replication fork while initiating the synthesis of Okazaki fragments on the lagging strand. We have determined a 3.45 A crystal structure of the T7 primase-helicase that shows an articulated arrangement of the primase and helicase sites. The crystallized primase-helicase is a heptamer with a crown-like shape, reflecting an intimate packing of helicase domains into a ring that is topped with loosely arrayed primase domains. This heptameric isoform can accommodate double-stranded DNA in its central channel, which nicely explains its recently described DNA remodeling activity. The double-jointed structure of the primase-helicase permits a free range of motion for the primase and helicase domains that suggests how the continuous unwinding of DNA at the replication fork can be periodically coupled to Okazaki fragment synthesis.

Bacteriophage T7↗
PubMed2002

Attenuating functions of the C terminus of lambda integrase.

The tyrosine family site-specific recombinases, in contrast to the related type I topoisomerases, which act as monomers on a single DNA molecule, rely on multi-protein complexes to synapse partner DNAs and coordinate two sequential strand exchanges involving four nicking-closing reactions. Here, we analyze three mutants of the catalytic domain of lambda integrase (Int), A241V, I353M and W350ter that are defective for normal recombination, but possess increased topoisomerase activity. The mutant enzymes can carry out individual DNA strand exchanges using truncated substrates or Holliday junctions, and they show more DNA-cleavage activity than wild-type Int on isolated att sites. Structural modeling predicts that the substituted residues may destabilize interactions between the C-terminal beta-strand (beta7) of Int and the core of the protein. The cleavage-competent state of Int requires the repositioning of the nucleophile (Y342) located on beta6 and the catalyst K235 located on the flexible beta2-beta3 loop, relative to their positions in a crystal structure of the inactive conformation. We propose that the anchoring of beta7 against the protein core restrains the movement of Tyr342 and/or Lys235, causing an attenuation of cleavage activity in most contexts. Within a bona fide recombination complex, the release of strand beta7 would allow Tyr342 and Lys235 to assume catalytically active conformations in coordination with other Int protomers in the complex. The loss of beta7 packing by misalignment or truncation in the mutant proteins described here causes a loss of regulated activity, thereby favoring DNA cleavage activity in monomeric complexes and forfeiting the coordination of strand-exchange necessary for efficient recombination.

Amino Acid Sequence↗
PubMed2002

Differential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase.

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.

Amino Acid Sequence↗