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Dorothy A Erie

Publications and source records attributed to Dorothy A Erie.

17 recordsLinked to original sources
PubMed2006

Unactivated PKR exists in an open conformation capable of binding nucleotides.

The dsRNA-activated protein kinase, PKR, plays a pivotal role in the cellular antiviral response. PKR contains an N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain. An autoinhibition model has been proposed in which latent PKR exists in a closed conformation where the substrate binding cleft of the kinase is blocked by the dsRBD. Binding to dsRNA activates the enzyme by inducing an open conformation and enhancing dimerization. We have tested this model by characterizing the affinity and kinetics of binding of a nucleotide substrate to PKR. The fluorescent nucleotide mant-AMPPNP binds to unactivated PKR with a Kd of approximately 30 microM, and the affinity is not strongly affected by autophosphorylation or binding to dsRNA. We observe biphasic binding kinetics in which the fast phase depends on ligand concentration but the slow phase is ligand-independent. The kinetic data fit to a two-step model of ligand binding followed by a slow conformation change. The kinetics are also not strongly affected by phosphorylation state or dsRNA binding. Thus, the equilibrium and kinetic data indicate that the substrate accessibility of the kinase is not modulated by PKR activation state as predicted by the autoinhibition model. In atomic force microscopy images, monomers of the latent protein are resolved with three separate regions linked by flexible, bridgelike structures. The resolution of the individual domains in the images supports a model in which unactivated PKR exists in an open conformation where the kinase domain is accessible and capable of binding substrate.

Adenylyl Imidodiphosphate↗
PubMed2006

Kinetic investigation of Escherichia coli RNA polymerase mutants that influence nucleotide discrimination and transcription fidelity.

Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue betaAsp(675), which is >10A away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues betaArg(678) and betaAsp(814) do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the betaAsp(675) mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.

Amino Acid Sequence↗
PubMed2006

UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.

Adenosine Triphosphatases↗
PubMed2005

Crystal structure of Thermus aquaticus Gfh1, a Gre-factor paralog that inhibits rather than stimulates transcript cleavage.

Transcription elongation in bacteria is promoted by Gre-factors, which stimulate an endogenous, endonucleolytic transcript cleavage activity of the RNA polymerase. A GreA paralog, Gfh1, present in Thermus aquaticus and Thermus thermophilus, has the opposite effect on elongation complexes, inhibiting rather than stimulating transcript cleavage. We have determined the 3.3 angstroms-resolution X-ray crystal structure of T.aquaticus Gfh1. The structure reveals an N-terminal and a C-terminal domain with close structural similarity to the domains of GreA, but with an unexpected conformational change in terms of the orientation of the domains with respect to each other. However, structural and functional analysis suggests that when complexed with RNA polymerase, Gfh1 adopts a conformation similar to that of GreA. These results reveal considerable structural flexibility for Gfh1, and for Gre-factors in general, as suggested by structural modeling, and point to a possible role for the conformational switch in Gre-factor and Gfh1 regulation. The opposite functional effect of Gfh1 compared with GreA may be determined by three structural characteristics. First, Gfh1 lacks the basic patch present in Gre-factors that likely plays a role in anchoring the 3'-fragment of the back-tracked RNA. Second, the loop at the tip of the N-terminal coiled-coil is highly flexible and contains extra acidic residues compared with GreA. Third, the N-terminal coiled-coil finger lacks a kink in the first alpha-helix, resulting in a straight coiled-coil compared with GreA. The latter two characteristics suggest that Gfh1 chelates a magnesium ion in the RNA polymerase active site (like GreA) but in a catalytically inactive configuration.

Amino Acid Sequence↗
PubMed2005

Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions.

Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein-DNA complexes and determining the stoichiometries and affinities of protein-protein complexes. We extend the capabilities of AFM to the determination of protein-DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein-DNA complexes.

Adenosine Triphosphatases↗
PubMed2005

AFM studies on the role of the protein RdgC in bacterial DNA recombination.

Genetic studies of rdgC in different bacterial systems suggest that it may play a role in replication and recombination. However, the exact function of the corresponding protein, RdgC, is unknown. In this study, we have imaged complexes of RdgC with both linear and supercoiled circular plasmid DNA using atomic force microscopy. We confirm that RdgC does not target any specific sequences in double-stranded DNA, as has been suggested from biochemical data. However, we detect an increased affinity of the protein to DNA ends, and an ability to promote bending of DNA. Similar binding preferences have been reported for enzymes involved in recombination. Protein complexes with supercoiled plasmid DNA further enabled us to study the effect of RdgC on DNA superstructure. At high concentrations of protein we observed promotion of DNA condensation. Recombination is largely enhanced by close contacts of distant regions along the DNA strands, as can occur, for instance, through condensation. Our data thus support a possible function of RdgC as a midwife of recombination.

DNA, Bacterial↗
PubMed2005

DNA mismatch repair.

DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication and escape proofreading. MMR proteins also participate in many other DNA transactions, such that inactivation of MMR can have wide-ranging biological consequences, which can be either beneficial or detrimental. We begin this review by briefly considering the multiple functions of MMR proteins and the consequences of impaired function. We then focus on the biochemical mechanism of MMR replication errors. Emphasis is on structure-function studies of MMR proteins, on how mismatches are recognized, on the process by which the newly replicated strand is identified, and on excision of the replication error.

Adenosine Triphosphatases↗
PubMed2003

DNA bending and unbending by MutS govern mismatch recognition and specificity.

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy. Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity.

Adenosine Triphosphatases↗
PubMed2003

Downstream DNA sequence effects on transcription elongation. Allosteric binding of nucleoside triphosphates facilitates translocation via a ratchet motion.

The ability of RNA polymerase (RNAP) to adopt multiple conformations is central to transcriptional regulation. In previous work, we demonstrated that RNAP can exist in an unactivated state that catalyzes synthesis slowly and an activated state that catalyzes synthesis rapidly, with the transition from the unactivated to the activated state being induced by the templated NTP binding to an allosteric site on the RNAP. In this work, we investigate the effects of downstream DNA sequences on the kinetics of single nucleotide incorporation. We demonstrate that changing the identity of the DNA base 1 bp downstream (+2) from the site of incorporation (+1) can regulate the catalytic activity of RNAP. Combining these data with sequence and structural analyses and molecular modeling, we identify the streptolydigin-binding region (Escherichia coli beta residues 543-546), which lies across from the downstream DNA, as the putative allosteric NTP binding site. We present a structural model in which the NTP binds to the streptolydigin loop and upon pairing with the +1 DNA base in the unactivated state or the +2 DNA base in the activated state facilitates translocation via a ratchet motion. This model provides an alternative mechanism for pausing as well as a structural explanation not only for our kinetic data but also for data from elongation studies on yeast RNAP II.

Allosteric Regulation↗
PubMed2003

Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.

Atomic force microscopy (AFM) has been applied in many biological investigations in the past 15 years. This review focuses on the application of AFM for quantitatively characterizing the structural and thermodynamic properties of protein-protein and protein-nucleic acid complexes. AFM can be used to determine the stoichiometries and association constants of multiprotein assemblies and to quantify changes in conformations of proteins and protein-nucleic acid complexes. In addition, AFM in solution permits the observation of the dynamic properties of biomolecular complexes and the measurement of intermolecular forces between biomolecules. Recent advances in cryogenic AFM, AFM on two-dimensional crystals, carbon nanotube probes, solution imaging, high-speed AFM, and manipulation capabilities enhance these applications by improving AFM resolution and the dynamic and operative capabilities of the AFM. These developments make AFM a powerful tool for investigating the biomolecular assemblies and interactions that govern gene regulation.

Air↗
PubMed2002

Functional oligomeric state of avian sarcoma virus integrase.

Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the avian sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.

Antiviral Agents↗
PubMed2002

The many conformational states of RNA polymerase elongation complexes and their roles in the regulation of transcription.

Transcription is highly regulated both by protein factors and by specific RNA or DNA sequence elements. Central to this regulation is the ability of RNA polymerase (RNAP) to adopt multiple conformational states during elongation. This review focuses on the mechanism of transcription elongation and the role of different conformational states in the regulation of elongation and termination. The discussion centers primarily on data from structural and functional studies on Escherichia coli RNAP. To introduce the players, a brief introduction to the general mechanism of elongation, the regulatory proteins, and the conformational states is provided. The role of each of the conformational states in elongation is then discussed in detail. Finally, an integrated mechanism of elongation is presented, bringing together the panoply of experiments.

Allosteric Site↗
PubMed2002

A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3'- 5' exonuclease and 5'-protruding strand endonuclease activities.

Werner syndrome is a rare autosomal recessive disease characterized by a premature aging phenotype, genomic instability, and a dramatically increased incidence of cancer and heart disease. Mutations in a single gene encoding a 1432-amino acid helicase/exonuclease (hWRN) have been shown to be responsible for the development of this disease. We have cloned, overexpressed, and purified a minimal, 171-amino acid fragment of hWRN that functions as an exonuclease. This fragment, encompassing residues 70-240 of hWRN (hWRN-N(70-240)), exhibits the same level of 3'-5' exonuclease activity as the previously described exonuclease fragment encompassing residues 1-333 of the full-length protein. The fragment also contains a 5'-protruding DNA strand endonuclease activity at a single-strand-double-strand DNA junction and within single-stranded DNA, as well as a 3'-5' exonuclease activity on single-stranded DNA. We find hWRN-N(70-240) is in a trimer-hexamer equilibrium in the absence of DNA when examined by gel filtration chromatography and atomic force microscopy. Upon addition of DNA substrate, hWRN-N(70-240) forms a hexamer and interacts with the recessed 3'-end of the DNA. Moreover, we find that the interaction of hWRN-N(70-240) with the replication protein PCNA also causes this minimal, 171-amino acid exonuclease region to form a hexamer. Thus, the active form of this minimal exonuclease fragment of human WRN appears to be a hexamer. The implications these results have on our understanding of hWRN's roles in DNA replication and repair are discussed.

Amino Acid Sequence↗
PubMed2002

DNA binding properties of the yeast Msh2-Msh6 and Mlh1-Pms1 heterodimers.

We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-Pms1, two protein complexes required to repair mismatches generated during DNA replication. Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA. The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity. These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-Pms1 heterodimer. Mlh1-Pms1 also binds to DNA, but independently of a mismatch. Mlh1-Pms1 binds short DNA substrates with low affinity and with a slight preference for single-stranded DNA. It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding. Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes. The novel DNA binding properties of Mlh1-Pms1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.

Base Pair Mismatch↗
PubMed2001

Transcript cleavage by Thermus thermophilus RNA polymerase. Effects of GreA and anti-GreA factors.

All known multisubunit RNA polymerases possess the ability to endonucleolytically degrade the nascent RNA transcript. To gain further insight into the conformational changes that govern transcript cleavage, we have examined the effects of certain anions on the intrinsic transcript cleavage activity of Thermus thermophilus RNA polymerase. Our results indicate that the conformational transitions involved in transcript cleavage, and therefore backtracking, are anion-dependent. In addition to characterizing the intrinsic cleavage activity of T. thermophilus RNA polymerase, we have identified, cloned, and expressed a homolog of the prokaryotic transcript cleavage factor GreA from the extreme thermophiles, T. thermophilus and Thermus aquaticus. The thermostable GreA factors contact the 3'-end of RNA, stimulate the intrinsic cleavage activity of T. thermophilus RNA polymerase, and increase the k(app) of the cleavage reaction 25-fold. In addition, we have identified a novel transcription factor in T. thermophilus and T. aquaticus that shares a high degree of sequence similarity with GreA, but has several residues that are not conserved with the N-terminal "basic patch" region of GreA. This protein, Gfh1, functions as an anti-GreA factor in vitro by reducing intrinsic cleavage and competing with GreA for a binding site on the polymerase.

Amino Acid Sequence↗