Thomas W. Kirby

Structure of the Escherichia coli DNA Polymerase III ϵ-HOT Proofreading Complex

The ϵ subunit of Escherichia coli DNA polymerase III possesses 3′-exonucleolytic proofreading activity. Within the Pol III core, ϵ is tightly bound between the α subunit (DNA polymerase) and θ subunit. Here, we present the crystal structure of ϵ in complex with HOT, the bacteriophage P1-encoded homolog of θ, at 2.1 Å resolution. The ϵ-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the ϵ central β-sheet as well as interactions between HOT and the catalytically important helix α1-loop-helix α2 motif of ϵ. This structure provides insight into how HOT and, by implication, θ may stabilize the ϵ subunit, thus promoting efficient proofreading during chromosomal replication.

Nuclear Magnetic Resonance Solution Structure of the Escherichia coli DNA Polymerase III θ Subunit

The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: α, e, and θ. The α subunit contains the polymerase, and the e subunit contains the exonucleolytic proofreading function. The small (8-kDa) θ subunit binds only to e. Its function is not well understood, although it was shown to exert a small stabilizing effect on the e proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, θ yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the θ homolog from bacteriophage P1, named HOT, we constructed a homology model of θ. This model suggested that the unfavorable behavior of θ might arise from exposed hydrophobic residues, particularly toward the end of α-helix 3. In gel filtration studies, θ elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded 1H-15N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of θ in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported θ structure. The new θ structure is expected to provide additional insight into its physiological role and its effect on the e proofreading subunit.

The Nuclease A Inhibitor Represents a New Variation of the Rare PR-1 Fold

Nuclease A (NucA) from Anabaena sp. is a non-specific endonuclease able to degrade single and double-stranded DNA and RNA. The endonucleolytic activity is inhibited by the nuclease A inhibitor (NuiA), which binds to NucA with 1:1 stoichiometry and picomolar affinity. In order to better understand the mechanism of inhibition, the solution structure of NuiA was determined by NMR methods. The fold of NuiA is an alpha-beta-alpha sandwich but standard database searches by DALI and TOP revealed no structural homologies. A visual inspection of alpha-beta-alpha folds in the CATH database revealed similarities to the PR-1-like fold (SCOP nomenclature). The similarities include the ordering of secondary structural elements, a single helix on one face of the alpha-beta-alpha sandwich, and three helices on the other face. However, a major difference is in the IV helix, which in the PR-1 fold is short and perpendicular to the I and III helices, but in NuiA is long and parallel to the I and III helices. Additionally, a strand insertion in the beta-sheet makes the NuiA beta-sheet completely antiparallel in organization. The fast time-scale motions of NuiA, characterized by enhanced flexibility of the extended loop between helices III and IV, also show similarities to P14a, which is a PR-1 fold. We propose that the purpose of the PR-1 fold is to form a stable scaffold to present this extended structure for biological interactions with other proteins. This hypothesis is supported by data that show that when NuiA is bound to NucA significant changes in chemical shift occur in the extended loop between helices III and IV.

Nuclear Localization of the DNA Repair Scaffold XRCC1: Uncovering the Functional Role of a Bipartite NLS

We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior provides a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex.

The ϵ subunit of Escherichia coli DNA polymerase III possesses 3′-exonucleolytic proofreading activity. Within the Pol III core, ϵ is tightly bound between the α subunit (DNA polymerase) and θ subunit. Here, we present the crystal structure of ϵ in complex with HOT, the bacteriophage P1-encoded homolog of θ, at 2.1 Å resolution. The ϵ-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the ϵ central β-sheet as well as interactions between HOT and the catalytically important helix α1-loop-helix α2 motif of ϵ. This structure provides insight into how HOT and, by implication, θ may stabilize the ϵ subunit, thus promoting efficient proofreading during chromosomal replication.

Substrate Rescue of DNA Polymerase β Containing a Catastrophic L22P Mutation

DNA polymerase (pol) β is a multidomain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single-strand break repair pathways. The high frequency of pol β variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making the determination of the functional consequences of these variants of interest. Pol β containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. Nuclear magnetic resonance (NMR) spectroscopic evaluations of both pol β and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca2+ and a complementary dNTP indicates that the fraction of pol β(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol β indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol β provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the “inactive” variant interferes with DNA repair.

Structure of the Escherichia coli DNA Polymerase III ε-HOT Proofreading Complex

The ϵ subunit of Escherichia coli DNA polymerase III possesses 3′-exonucleolytic proofreading activity. Within the Pol III core, ϵ is tightly bound between the α subunit (DNA polymerase) and θ subunit. Here, we present the crystal structure of ϵ in complex with HOT, the bacteriophage P1-encoded homolog of θ, at 2.1 Å resolution. The ϵ-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the ϵ central β-sheet as well as interactions between HOT and the catalytically important helix α1-loop-helix α2 motif of ϵ. This structure provides insight into how HOT and, by implication, θ may stabilize the ϵ subunit, thus promoting efficient proofreading during chromosomal replication.

DNA polymerase β contains a functional nuclear localization signal at its N-terminus

Abstract DNA polymerase β (pol β) requires nuclear localization to fulfil its DNA repair function. Although its small size has been interpreted to imply the absence of a need for active nuclear import, sequence and structural analysis suggests that a monopartite nuclear localization signal (NLS) may reside in the N-terminal lyase domain. Binding of this domain to Importin α1 (Impα1) was confirmed by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol β residues 2–13. These studies indicate high affinity binding, characterized by a low micromolar Kd, that is selective for the murine Importin α1 (mImpα1) minor site, with the Kd strengthening to ∼140 nM for the full lyase domain (residues 2–87). A further reduction in Kd obtains in binding studies with human Importin α5 (hImpα5), which in some cases has been demonstrated to bind small domains connected to the NLS. The role of this NLS was confirmed by fluorescent imaging of wild-type and NLS-mutated pol β(R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol β. Together these data demonstrate that pol β contains a specific NLS sequence in the N-terminal lyase domain that promotes transport of the protein independent of its interaction partners. Active nuclear uptake allows development of a nuclear/cytosolic concentration gradient against a background of passive diffusion.

Characterization of the Redox Transition of the XRCC1 N-terminal Domain

XRCC1, a scaffold protein involved in DNA repair, contains an N-terminal domain (X1NTD) that interacts specifically with DNA polymerase β. It was recently discovered that X1NTD contains a disulfide switch that allows it to adopt either of two metamorphic structures. In the present study, we demonstrate that formation of an N-terminal proline carbimate adduct resulting from the nonenzymatic reaction of Pro2 with CO2 is essential for stabilizing the oxidized structure, X1NTDox. The kinetic response of X1NTDred to H2O2, monitored by NMR, was determined to be very slow, consistent with involvement of the buried, kinetically trapped Cys12 residue, but was significantly accelerated by addition of protein disulfide isomerase or by Cu(2+). NMR analysis of a sample containing the pol β polymerase domain, and both the reduced and oxidized forms of X1NTD, indicates that the oxidized form binds to the enzyme 25-fold more tightly than the reduced form.

Metabolic transformation of AZTp4A by Ap4A hydrolase regenerates AZT triphosphate

The reverse transcriptase (RT) of HIV which has been inhibited by the incorporation of AZT into the primer strand is subject to a deblocking reaction by cellular ATP. This reaction yields unblocked primer plus the dinucleoside tetraphosphate, AZTp(4)A. In the present study, we report that AZTp(4)A is an excellent substrate for the enzyme Ap(4)A hydrolase (asymmetrical dinucleoside tetraphosphatase, EC 3.6.1.17), an enzyme that is widely distributed in many cell types. Progress of the reaction has been monitored by 31P NMR, and it was found that hydrolysis results in the production of AZTTP:ATP in a 7:1 ratio. The AZTp(4)A was also hydrolyzed at a rate 1.8-fold more rapidly than Ap(4)A. Spectrophotometric assays yielded Michaelis constants of 2.35 and 0.71 microM for Ap(4)A and AZTp(4)A, respectively. It, therefore, appears that Ap(4)A hydrolase can play a useful role in the regeneration of the AZTTP, the active form of AZT, for the inhibition of HIV RT.