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. DNA polymerase beta (pol beta) plays a key role in base excision repair, as well as participating in other repair pathways and in lesion bypass. Although its small size has been interpreted to imply the absence of a need for active nuclear import, sequence and structural analysis suggested that a monopartite nuclear localization signal (NLS) may reside in the N-terminal lyase domain. Binding of this domain to Importin alpha1 (Impalpha1) was demonstrated by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol beta residues 2 -13. These studies demonstrated a dissociation constant in the low micromolar range. Atypically, studies using Impalpha mutants further demonstrated that this interaction is selective for the minor binding pocket of Impalpha. Ligand competition studies demonstrated that the full lyase domain binds with higher affinity than the N-terminal peptide, with a Kd = 140 nM. Since the human cell contains additional Impalpha isoproteins, we also tested several others likely to mediate pol import, and determined that the interaction of impalpha5 with the pol lyase domain has a dissociation constant of 17 nM. The functional importance of this NLS was confirmed by fluorescent imaging of wild-type and NLS-mutated pol beta(R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol beta, which showed approximate equilibration of nuclear and cytosolic pol beta concentrations. Together these data demonstrate that pol beta contains a specific NLS sequence in the N-terminal lyase domain that promotes its active transport independent of its interaction partners. Active nuclear uptake allows development of a nuclear/cytosolic concentration gradient against a background of passive diffusion. Among other factors, active transport supports the availability of pol beta for other repair activities including DNA scanning and trans-lesion synthesis. Project 2. Aprataxin and PNKP-like factor (APLF) was recently identified as a novel component of non-homologous end-joining that promotes intracellular re-joining of transfected linear plasmid DNA molecules and accelerates the repair of chromosomal double strand breaks following gamma irradiation. The enzyme contains a forkhead associated (FHA) domain that competes with FHA domain-containing aprataxin and PNKP for a common phosphorylated FHA binding motif (FBM) on XRCC1 that is located immediately before the C-terminal BRCT domain. Although aprataxin and PNKP play important roles in preparing the ends of the broken DNA strand for subsequent enzymatic chemistry, APLF does not fulfill such a role, so that the function of the XRCC1-APLF interaction is unclear. During the past year, we characterized this interaction using NMR spectroscopy and X-ray crystallography. It was shown that the FHA binding site is highly selective for the dianionic form of the pT519 residue on the FBM. We also showed that the FBM on XRCC1 is characterized by unusually high phosphothreonine and phosphoserine pK values as a consequence of the highly anionic nature of the FBM peptide. Further, FBM-FHA binding specificity is dependent on a relatively short phosphopeptide motif, while binding affinity is determined by a much longer segment as a result of non-specific electrostatic interactions. Although pT519 interacts directly with the APLF FHA domain, pS518 extends away from the structure and does not interact directly, however this residue contributes significantly to the observed binding affinity. It was proposed that the role of pS518 is to act as a local buffer that facilitates protonation/deprotonation of pT519. Such buffer-dependent effects are important when the protonation state of the uncomplexed and protein-complexed peptide differ significantly. Project 3. Recent collaborative studies with the Williams group have involved DNA repair proteins APE2 and TDP2. AP endonuclease 2 participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via poorly understood mechanisms. During the past year it was shown that resection activity is regulated by DNA interactions in the Zf-GRF domain, a region sharing high homology with DNA damage response proteins Topoisomerase 3alpha and NEIL3 DNA glycosylase, as well as transcription and RNA regulatory proteins such as TTF2, TFIIS and RPB9. Biochemical and NMR studies established the nucleic acid binding activity of the Zf-GRF domain. Further, an APE2 Zf-GRF X-ray structure and small angle X-ray scattering (SAXS) analyses showed that the Zf-GRF fold adopts a crescent-shaped ssDNA binding claw, that is flexibly appended to an APE2 endonuclease/exonuclease/ phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impacted both DNA binding and 3'-5' exonuclease processing, and prevented efficient APE2-dependant RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress. In a collaborative study of tyrosyl-DNA phosphodiesterase 2 (TDP2), NMR was also used to evaluate the binding of TDP2 with SUMO2.