Targeting the Structural Maturation Pathway of HIV-1 Reverse Transcriptase.

Formation of active HIV-1 reverse transcriptase (RT) proceeds via a structural maturation process that involves subdomain rearrangements and formation of an asymmetric p66/p66' homodimer. These studies were undertaken to evaluate whether the information about this maturation process can be used to identify small molecule ligands that retard or interfere with the steps involved. We utilized the isolated polymerase domain, p51, rather than p66, since the initial subdomain rearrangements are largely limited to this domain. Target sites at subdomain interfaces were identified and computational analysis used to obtain an initial set of ligands for screening. Chromatographic evaluations of the p51 homodimer/monomer ratio support the feasibility of this approach. Ligands that bind near the interfaces and a ligand that binds directly to a region of the fingers subdomain involved in subunit interface formation were identified, and the interactions were further characterized by NMR spectroscopy and X-ray crystallography. Although these ligands were found to reduce dimer formation, further efforts will be required to obtain ligands with higher binding affinity. In contrast with previous ligand identification studies performed on the RT heterodimer, subunit interface surfaces are solvent-accessible in the p51 and p66 monomers, making these constructs preferable for identification of ligands that directly interfere with dimerization.

Phosphopeptide interactions of the Nbs1 N-terminal FHA-BRCT1/2 domains.

Human Nbs1, a component of the MRN complex involved in DNA double strand break repair, contains a concatenated N-terminal FHA-BRCT1/2 sequence that supports interaction with multiple phosphopeptide binding partners. MDC1 binding localizes Nbs1 to the damage site, while binding of CDK-phosphorylated CtIP activates additional ATM-dependent CtIP phosphorylation, modulating substrate-dependent resection. We have investigated the phosphopeptide binding characteristics of Nbs1 BRCT1/2 based on a molecular modeling approach that revealed structural homology with the tandem TopBP1 BRCT7/8 domains. Relevance of the model was substantiated by the ability of TopBP1-binding FANCJ phosphopeptide to interact with hsNbsBRCT1/2, albeit with lower affinity. The modeled BRCT1/2 is characterized by low pSer/pThr selectivity, preference for a cationic residue at the + 2 position, and an inter-domain binding cleft selective for hydrophobic residues at the + 3/ + 4 positions. These features provide insight into the basis for interaction of SDT motifs with the BRCT1/2 domains and allowed identification of CtIP pSer347- and pThr847-containing phosphopeptides as high and lower affinity ligands, respectively. Among other binding partners considered, rodent XRCC1 contains an SDT sequence in the second linker consistent with high-affinity Nbs1 binding, while human XRCC1 lacks this motif, but contains other phosphorylated sequences that exhibit low-affinity binding.

Ligand binding characteristics of the Ku80 von Willebrand domain.

The N-terminal von Willebrand domain of Ku80 supports interactions with a Ku binding motif (KBM) that has been identified in at least three other DNA repair proteins: the non-homologous end joining (NHEJ) scaffold APLF, the modulator of retrovirus infection, MRI, and the Werner syndrome protein (WRN). A second, more recently identified Ku binding motif present in XLF and several other proteins (KBMX) has also been reported to interact with this domain. The isolated Ku80 von Willebrand antigen domain (vWA) from Xenopus laevis has a sequence that is 60% identical with the human domain, is readily expressed and has been used to investigate these interactions. Structural characterization of the complexes formed with the KBM motifs in human APLF, MRI, and WRN identify a conserved binding site that is consistent with previously-reported mutational studies. In contrast with the KBM binding site, structural studies indicate that the KBMX site is occluded by a distorted helix. Fluorescence polarization and 19F NMR studies of a fluorinated XLF C-terminal peptide failed to indicate any interaction with the frog vWA. It was hypothesized that availability of this binding site is conditional, i.e., dependent on specific experimental conditions or other repair factors to make the site available for binding. Modulating the fraction of KBMX-accessible binding site mutationally demonstrated that the more open site is capable of binding the KBMXXLF motif peptide. It is suggested that the conditional nature of KBMX binding limits formation of non-productive complexes so that activation-dependent site availability can more optimally support advancing the synapsis process.

Variations in nuclear localization strategies among pol X family enzymes.

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional nuclear localization signal (NLS) in DNA polymerase ß, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase mu (pol µ) and DNA polymerase lambda (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol µ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol µ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase.

Transitions in DNA polymerase ß µs-ms dynamics related to substrate binding and catalysis.

DNA polymerase ß (pol ß) plays a central role in the DNA base excision repair pathway and also serves as an important model polymerase. Dynamic characterization of pol ß from methyl-TROSY 13C-1H multiple quantum CPMG relaxation dispersion experiments of Ile and Met sidechains and previous backbone relaxation dispersion measurements, reveals transitions in µs-ms dynamics in response to highly variable substrates. Recognition of a 1-nt-gapped DNA substrate is accompanied by significant backbone and sidechain motion in the lyase domain and the DNA binding subdomain of the polymerase domain, that may help to facilitate binding of the apoenzyme to the segments of the DNA upstream and downstream from the gap. Backbone µs-ms motion largely disappears after formation of the pol ß-DNA complex, giving rise to an increase in uncoupled µs-ms sidechain motion throughout the enzyme. Formation of an abortive ternary complex using a non-hydrolyzable dNTP results in sidechain motions that fit to a single exchange process localized to the catalytic subdomain, suggesting that this motion may play a role in catalysis.

Characterization of the APLF FHA-XRCC1 phosphopeptide interaction and its structural and functional implications.

Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHA-FBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr. These high pK values are characteristic of the polyanionic peptides typically produced by CK2 phosphorylation. Binding affinity is greatly enhanced by residues flanking the crystallographically-defined recognition motif, apparently as a consequence of non-specific electrostatic interactions, supporting the role of XRCC1 in nuclear cotransport of APLF. The FHA domain-dependent interaction of XRCC1 with APLF joins repair scaffolds that support single-strand break repair and non-homologous end joining (NHEJ). It is suggested that for double-strand DNA breaks that have initially formed a complex with PARP1 and its binding partner XRCC1, this interaction acts as a backup attempt to intercept the more error-prone alternative NHEJ repair pathway by recruiting Ku and associated NHEJ factors.

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

DNA polymerase ß (pol ß) 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 ß 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 ß(R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol ß. Together these data demonstrate that pol ß 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.

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 ß binding domain. This behavior provides a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.

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 ß. 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 ß 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.

Substrate rescue of DNA polymerase ß containing a catastrophic L22P mutation.

DNA polymerase (pol) ß 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 ß 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 ß 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 ß 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 Ca(2+) and a complementary dNTP indicates that the fraction of pol ß(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 ß 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 ß 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.

Metal-induced DNA translocation leads to DNA polymerase conformational activation.

Binding of the catalytic divalent ion to the ternary DNA polymerase ß/gapped DNA/dNTP complex is thought to represent the final step in the assembly of the catalytic complex and is consequently a critical determinant of replicative fidelity. We have analyzed the effects of Mg(2+) and Zn(2+) on the conformational activation process based on NMR measurements of [methyl-(13)C]methionine DNA polymerase ß. Unexpectedly, both divalent metals were able to produce a template base-dependent conformational activation of the polymerase/1-nt gapped DNA complex in the absence of a complementary incoming nucleotide, albeit with different temperature thresholds. This conformational activation is abolished by substituting Glu295 with lysine, thereby interrupting key hydrogen bonds necessary to stabilize the closed conformation. These and other results indicate that metal-binding can promote: translocation of the primer terminus base pair into the active site; expulsion of an unpaired pyrimidine, but not purine, base from the template-binding pocket; and motions of polymerase subdomains that close the active site. We also have performed pyrophosphorolysis studies that are consistent with predictions based on these results. These findings provide new insight into the relationships between conformational activation, enzyme activity and polymerase fidelity.

NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution.

UvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. In order to gain insight into the behavior of UvrB in solution, we have performed NMR studies on [methyl-13C]methionine-labeled UvrB from Bacillus caldotenax (molecular mass=75 kDa). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). M632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer-dimer exchange and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB-domain 4 heterodimer. A dissociation constant (KD) value of 3.3 microM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3-4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment but by direct interactions with domains 1-3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB-domain 4 heterodimerization are allowed.

NMR assignment of polymerase beta labeled with 2H, 13C, and 15N in complex with substrate DNA.

DNA polymerase beta is a multifunctional enzyme involved in base excision repair of nuclear DNA invertebrate cells. We present here the first assignments of the full-length protein (335 residues, 39 kDa) in the presence of a double gap--double hairpin DNA (22 nucleotides, 7 kDa).

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

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.

NMR determination of lysine pKa values in the Pol lambda lyase domain: mechanistic implications.

The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase beta (Pol beta), and for the homologous domain of DNA polymerase lambda (Pol lambda). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol lambda), followed by a beta-elimination reaction. To better understand the underlying chemistry, we have determined pKa values for the lysine residues in the Pol lambda lyase domain labeled with [epsilon-13C]lysine. At neutral pH, the H(epsilon) protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pKa value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of approximately 1:1.3, consistent with a dissociation constant of approximately 1 microM. The epsilon-proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pKa values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general increase in the Hill coefficients observed in the complex is consistent with the screening of the interacting lysine residues by the DNA. The pKa of K312 residue increased to 10.58 in the complex, probably due to salt bridge formation with the 5'-phosphate group of the DNA. The pKa values obtained for the lyase domain of Pol lambda in the present study are consistent with recent crystallographic studies of Pol beta complexed with 5-phosphorylated abasic sugar analogues in nicked DNA which reveal an open site with no obvious interactions that would significantly depress the pK value for the active site lysine residue. It is suggested that due to the heterogeneity of the damaged DNA substrates with which Pol lambda as well as other related polymerases may be required to bind, the unexpectedly poor optimization of the lyase catalytic site may reflect a compromise of flexibility with catalytic efficiency.

A thymine isostere in the templating position disrupts assembly of the closed DNA polymerase beta ternary complex.

The high fidelity of the DNA polymerization process is critically important for the stability of the cellular genome. The role of template and incoming nucleotide base pairing in polymerase fidelity has recently been explored by the use of nucleotide isosteres, which preserve the steric but not the electronic properties of the corresponding bases. The DNA repair enzyme, DNA polymerase beta (Pol beta), is among the most discriminating, being inactive when the thymine isostere difluorotoluene (DFT) is present in the templating base position. To explore the physical basis for this inactivity, we have performed NMR studies on [methyl-13C]methionine-labeled Pol beta complexed with double-hairpin DNA, used to model the gapped nucleotide substrate, and having either a thymine or a DFT isostere at the templating base position. The six methionine residues distributed throughout the enzyme provide useful conformational probes of the lyase and polymerase domains and subdomains. Analysis of the proton shift of Met282 that results from formation of an abortive Pol beta-gapped DNA-dATP complex is consistent with an open to closed conformational change of the enzyme predicted from crystal structures. In contrast, the same resonance is nearly unshifted when a ternary complex is formed from dATP and gapped DNA in which a DFT isostere replaces thymine at the templating base position. Alternatively, the resonances of Met191 and Met155, located in the catalytic subdomain, show perturbations upon formation of the abortive ternary complex, which are qualitatively similar, but significantly weaker, than the changes observed when thymine is present at the templating base position. The changes in the Met155 and Met191 methyl resonances are in fact more similar to those observed in the binary Pol beta-dATP complex. These studies demonstrate that the block in catalysis is directly related to the absence of the set of conformational transitions that include the "open" to "closed" transition monitored by Met282.

Nuclear magnetic resonance solution structure of the Escherichia coli DNA polymerase III theta subunit.

The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: alpha, epsilon, and theta. The alpha subunit contains the polymerase, and the epsilon subunit contains the exonucleolytic proofreading function. The small (8-kDa) theta subunit binds only to epsilon. Its function is not well understood, although it was shown to exert a small stabilizing effect on the epsilon proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, theta yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the theta homolog from bacteriophage P1, named HOT, we constructed a homology model of theta. This model suggested that the unfavorable behavior of theta might arise from exposed hydrophobic residues, particularly toward the end of alpha-helix 3. In gel filtration studies, theta 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 theta in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported theta structure. The new theta structure is expected to provide additional insight into its physiological role and its effect on the epsilon proofreading subunit.

Phage like it HOT: solution structure of the bacteriophage P1-encoded HOT protein, a homolog of the theta subunit of E. coli DNA polymerase III.

DNA polymerase III, the main replicative polymerase of E. coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions. It was recently discovered that E. coli bacteriophage P1 encodes a theta homolog, named HOT. The (1)H-(15)N HSQC spectrum of HOT exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a determination of the HOT solution structure by NMR. The structure contains three alpha helices, as reported previously for theta; however, the folding topology of the two proteins is very different. Residual dipolar coupling measurements on labeled theta support the conclusion that it is structurally homologous with HOT. As judged by CD measurements, the melting temperature of HOT was 62 degrees C, compared to 56 degrees C for theta, consistent with other data suggesting greater thermal stability of the HOT protein.

Backbone dynamics of the RNase H domain of HIV-1 reverse transcriptase.

Previous NMR relaxation studies of the isolated RNase H domain of HIV-1 reverse transcriptase at low pH have revealed that it is substantially more dynamic and less ordered than the relatively stable and catalytically active E. coli RNase HI. Using more recently developed techniques, we have investigated the dynamic behavior of the RNase H domain of HIV-1 reverse transcriptase at a more physiological pH (6.8), under a variety of solution conditions: no Mg(2+), 80 mM Mg(2+), and 80 mM Mg(2+) plus AMP ligand. In addition, we have repeated the previous measurements on a sample containing 100 mM sodium acetate, pH 5.4. Under all conditions studied, the order parameters from NMR relaxation analysis are uniformly high (>0.8) for most of the domain with the exception of the C-terminal region. Subtle differences can be found among the conditions studied, although the statistical significance of the differences is marginal. Residues 71-114 show a slight increase in order parameter with the addition of 5'-AMP. Conformational exchange, measured with CPMG relaxation dispersion experiments in the presence of Mg and AMP, were detected for some NH sites, predominantly located in the N-terminal region of the protein near strands beta2 and beta3 and helix alpha(A) (residues 28-69). In contrast with earlier studies indicating pathologically extreme dynamic behavior that apparently correlated with inactivity of the isolated domain, the relaxation analysis under the conditions of the present study yielded parameters that are more similar to those of the active E. coli RNase HI. A comparison of the order parameters obtained from a model-free analysis of the relaxation data with the B-factors in the crystal structures of the RNase H domain, both for the isolated domain and for the full HIV-1 reverse transcriptase structure, suggests that the dynamic behavior is similar in all cases.

Dynamic characterization of a DNA repair enzyme: NMR studies of [methyl-13C]methionine-labeled DNA polymerase beta.

Crystallographic characterization of DNA polymerase beta (pol beta) has suggested that multiple-domain and subdomain motions occur during substrate binding and catalysis. NMR studies of [methyl-(13)C]methionine-labeled pol beta were conducted to characterize the structural and dynamic response to ligand binding. The enzyme contains seven methionine residues, one of which is at the amino terminus and is partially removed by the expression system. Three of the methyl resonances were readily assigned using site-directed mutants. Assignment of the resonances of Met155, Met158, and Met191 was more difficult due to the spatial proximity of these residues, so that assignments were based on NOESY-HSQC data and on the response to paramagnetic Co(2+) addition, as well as shift perturbations observed for the site-directed mutants. The response of the methyl resonances to substrate binding was evaluated by the serial addition of a template oligonucleotide, a downstream 5'-phosphorylated oligonucleotide, and a primer oligonucleotide to create a two-nucleotide-gapped DNA substrate. Addition of the single-stranded template DNA resulted in selective broadening of the methyl resonance of Met18 in the 8 kDa lyase domain, and this resonance then shifted and sharpened upon addition of a 5'-phosphate-terminated downstream complementary oligonucleotide. Conversion of the two-nucleotide-gapped DNA substrate to a single-nucleotide-gapped substrate by incorporation of ddCMP produced a small perturbation of the Met236 resonance, which makes contact with the primer strand in the crystal structure. The addition of a second equivalent of ddCTP to form the pol beta-DNA-ddCTP ternary complex resulted in significant shifts for the resonances corresponding to Met155, Met191, Met236, and Met282. The Met155 methyl resonance is severely broadened, while the Met191 and Met282 resonances exhibit significant but less extreme broadening. Since only Met236 makes contact with the substrate, the effects on Met155, Met236, and Met282 result from indirect conformational and dynamic perturbations. Previous crystallographic characterization of this abortive complex indicated that a polymerase subdomain or segment (alpha-helix N) repositions itself to form one face of the binding pocket for the nascent base pair. Met282 serves as a probe for motion in this segment. Addition of Mg(2+)-dATP to pol beta in the absence of DNA produced qualitatively similar but much smaller effects on Met191 and Met155, but did not strongly perturb Met282, leading to the conclusion that Mg(2+)-dATP alone is insufficient to produce the large conformational changes that are observed in the abortive complex involving the gapped DNA with a blocked primer and ddNTP. Thus, the NMR data indicate that the nucleotide-DNA interaction appears to be essential for conformational activation.