Nmr Studies Of Biomolecular Structure, Function, And Dynamics

National Institute Of Environmental Health Sciences

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Abstract

This project utilizes state-of-the-art NMR spectroscopy to characterize DNA damage and to determine the structure of enzymes involved in base excision repair and the intact base excision repair complex. The primary emphasis during the recent review period includes 1) structural characterization of the repair complex formed from the XRCC1 N-terminal domain and DNA pol beta;2) characterization of the interaction of XRCC1 BRCT domains with cognate BRCT domains on Ligase IIIa and on Poly(ADP-ribose) polymerase-1 (PARP-1);3) characterization of the structure of DNA containing isolated ribonucleotides;4) understanding the relationship of conformational activation of Pol beta to polymerase fidelity Project 1. Base excision repair (BER) is a complex process requiring the coordinated activities of multiple enzymes that interpret the damage, excise the damaged components, and resynthesize the DNA. Current understanding of the BER repair complex involves formation of transient complexes with the damaged DNA. These repair complexes are organized by the scaffold protein, XRCC1. However, the complex formed between the C-terminal BRCT domains of XRCC1 and Ligase IIIalpha is constitutive. Although the Ligase IIIalpha was the first XRCC1 binding partner to be identified, characterization of the underlying complex has eluded structural determination. Furthermore, all existing data for BRCT domain-mediated protein-protein complexes involved pairs of tandem BRCT domains, while this is not the case for the XRCC1-LigaseIIIalpha complex. In the absence of structural data, alternative proposals for this interaction have been advanced. Interpretation of these models is complicated by the formation of homodimers which, depending on the model, may either contribute to, or compete with heterodimer formation. During the past year, we determined the structures of both the X1BRCTb and the L3BRCT homodimers, as well as the heterodimeric X1BRCTb:L3BRCT complex. The initial structural characterization of the heterodimer suggested that additional residues might be involved in complex formation. On this basis, we studied a longer X1BRCTb construct, containing residues comprising the interdomain linker region immediately preceding X1BRCTb. Structural characterization of this extended construct revealed a structural motif which leads to significant stabilization of the heterodimer and dictates heterodimer/homodimer selectivity. The role of the additional linker residues was further confirmed by melting studies performed using circular dichroism. These data provide important fundamental insights into the structural basis of BRCT-mediated dimerization events. Additionally, the data resolve questions related to the organization of this critically important DNA repair complex. Project 2. We have also continued our studies of the interaction of the N-terminal domain of XRCC1 with the repair polymerase, Pol beta. We previously had determined that the N-terminal domain of XRCC1, X1NTD, is able to adopt two dramatically different structures, related to the presence or absence of a disulfide bond. In order to better understand the physiological role of this reversible disulfide bond formation, we have worked to better define the conditions that lead to the oxidized form of X1NTD. Our previous study had identified a mutation, I4D, that helped to stabilize the oxidized form. More recent studies have identified other experimental conditions that also stabilize this form of the protein. Based on the analysis of the backbone shifts using TALOS, we have been able to demonstrate that the solution structure obtained under these conditions is similar to that observed in the crystalline complex with Pol beta. Nevertheless, in order to understand the physiological significance of the oxidized form of X1NTD, it is necessary to further stabilize this form. We are currently exploring various mutational strategies that may provide a more succeessful degree of stabilization than the initial mutation used in our first study, without altering the electrostatic surface of the protein as significantly as the I4D substitution. Project 3. We have continued our evaluation of the effects of metals on DNA pol beta by studying the conformational response to divalent zinc. Binding of the catalytic divalent ion to the ternary DNA pol beta/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 Mg2+ and Zn2+ on the conformational activation process based on NMR measurements of methyl 13Cmethionine-labeled DNA polymerase beta. Unexpectedly, both divalent metals were able to produce a template base-dependent conformational activation of the polymerase/one-nucleotide gapped DNA complex in the absence of a complementary incoming nucleotide. This conformational activation can be abolished by substituting Glu295 with lysine, thereby interrupting key hydrogen bonds necessary to stabilize the closed conformation. These and other results suggest that metal-binding is able to 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. These results have important implications for fidelity and pyrophosphorolysis. Project 4. This project is directed at characterizing the interaction of the first XRCC1 BRCT domain, X1BRCTa, with its previously identified binding partner, PARP1. Although previous publications had indicated that X1BRCTa binds to the BRCT domain of PARP1, we find that, after an extensive series of studies, there is no significant binding affinity between the two BRCT domains. One possible resolution of this inconsistency is that the presence of poly(ADP-ribose) modification of the residues adjacent to the PARP1 BRCT domain is essential to obtain binding. We are currently evaluating this possibility. Project 5. Effect of 2'-hydroxylation on DNA structure. It has recently been demonstrated that the extent of incorporation of ribonucleotides by replicative polymerases is sufficient to make 2'-hydroxylation the most common mutation in newly synthesized DNA. We have used the Dickerson dodecamer as a model for the analysis of the effect of an isolated ribonucleotide on DNA structure. The self-complementary sequence investigated, d(CGC)rGd(TTAAGCGC), contains two symmetrically positioned guanidine residues paired with deoxycytidine residues. Consistent with previous studies of mixed ribo/deoxyribo nucleic acids, the absence of an observable H1'-H2'scalar coupling interaction is indicative of a C3'-endo conformation for the cytidine residue. However, longer range structural perturbations resulting from the presence of the ribonucleotide appear to be quite modest, so that the B-form DNA structure is maintained. During the past year we performed a more thorough analysis of complete structure of our model nucleotide, and also performed extensive molecular modeling calculations in order to more fully evaluate these results, and to extrapolate the conclusions to DNA containing a higher density of ribonucleotide substitutions.