Nmr Studies Of Biomolecular Structure, Function, And Dynamics

National Institute Of Environmental Health Sciences

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Abstract

Project 1. Efficient DNA repair is dependent on the intranuclear assembly of damage-dependent repair complexes, and thus requires the necessary DNA repair polymerases to be present in the cell nucleus. The linkage between various diseases and impairment of nuclear localization is increasingly being substantiated. We had previously found that, in contrast with the generally held belief, DNA pol contains a classical nuclear localization sequence (NLS) at its N-terminus. We subsequently decided to investigate the putative NLS signal for the remaining pol X family enzymes: Terminal deoxynucleotidyl transferase (TdT), DNA polymerase mu, and DNA polymerase lambda. Among these three enzymes, TdT has an easily identified NLS that scores highly using any of a number of NLS prediction programs. Alternatively, none of the six different programs that we tried identified high scoring NLS signals for pol mu or pol lambda. The NLS sequences for each polymerase were identified based on Importin alpha (Impalpha) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impalpha complexes and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol mu utilize monopartite NLS sequences, while pol lambda utilizes a bipartite sequence, unique among the pol X family members. The pol mu NLS has relatively low affinity for Importin alpha, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS with the Impalpha binding pockets. For pol mu, 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. Pol lambda utilizes a non-canonical bipartite NLS sequence, so that the major site motif contains only two basic residues, using an isoleucine at position P3. This type of substitution is, however, found in an increasing number of examples and is sufficient to support high affinity binding. Subcellular localization was evaluated using GFP-adducts of each polymerase, and the identities of the NLS residues were confirmed with mutational studies. Although the results of these studies were generally consistent with previous literature reports, the results for TdT localization differed from those of the Yale group (Repasky et al., J. Immunol. 172, 5478-5488; 2004), who reported that the putative NLS is not required for nuclear localization of TdT. Project 2. 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 FHAFBM 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), and thus apparently competing with the objectives of each scaffold-supported repair process. It was suggested that this overlapping interaction results in competitive repair pathways that compete to optimize the fidelity of the resulting repair, as has been discussed by Iliakis and coworkers. Project 3. DNA ligation is a central process in biology that finalizes genome maintenance metabolic processes including DNA replication, recombination, and DNA repair. Eukaryotic DNA ligases catalyze ligation via a three-step, ATP-dependent reaction. First, the DNA ligase active site lysine is adenylated. Second, the adenylate is transferred to a DNA 5' phosphate to facilitate the nick-sealing step. Third, nucleophilic attack of a 3'-OH on the activated 5'-adenylate facilitates phosphodiester bond formation, and sealing of the DNA break. Environmental and metabolic sources of DNA damage can result in abortive ligation, due to the failure to complete step 3, with the resulting generation of 5'-adenylated (5'-AMP) DNA strand breaks. The aprataxin (APTX) RNA-DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder ataxia with oculomotor ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. We have determined structures of APTX engaging nicked RNA-DNA substrates that provide direct evidence for a wedge-pivot-cut strategy for 5'-AMP resolution shared with the alternate 5'-AMP processing enzymes DNA polymerase beta and flap endonuclease 1 (FEN1). These studies reveal a DNA-induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. We also have defined a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and unexpectedly, a dominant AOA1 variant allosterically modulates APTX active site conformations. As noted above, aprataxin contains a histidine triad (HIT) nucleotide hydrolase whose function can be perturbed by mutations that have been associated with patients afflicted with AOA1. We recently initiated a further, more detailed NMR investigation of the behavior of the catalytic histidines of the enzyme aimed at better understanding the molecular basis for functional impairment by many of these reported mutations. As is often the case, the basis for functional impairment by some of these mutations is straightforward, while the mechanism by which other mutations interfere with catalytic function is much less obvious. It is anticipated that these additional studies will provide a more detailed and sensitive basis for understanding the effects of these additional mutations. Project 4. Folate metabolism plays a central role in nucleotide biosynthesis and its perturbation can be mutagenic. More generally, dysregulation of folate metabolism appears to be broadly linked to deficiencies in genome methylation, stability and repair. Since biguanide compounds have been developed as inhibitors of folate metabolizing enzymes such as dihydrofolate reductase (DHFR), and since this class of compounds is widely prescribed to treat Type II diabetes, we have investigated whether they might also interfere with folate metabolism generally and, more specifically, with the function of DHFR. We found that the biguanide drug phenformin is a weak inhibitor of the E. coli DHFR (Ki 200 microM), with the more popularly prescribed drug metformin being substantially weaker. However, the folate binding site can be considered to be composed of a pteridine binding subsite to which the biguanides bind, as well as a p-aminobenzoylglutamate (pABG) subsite to which other compounds can bind. Most recently, we have investigated the binding of various NSAIDs to the pABG subsite, and cooperative binding of lignds to both sites.