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 (PARP1); and 3) characterization of the structure of DNA containing isolated ribonucleotides. Project 1. The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase3alpha. For efficient ligation, ligase3alpha is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available for some time, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced for the specific interactions, including the proposal that the complex involves an interaction between two BRCT homodimers to form a tetrameric protein complex. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We have been able to observe and structurally characterize the Ligase3alpha-XRCC1 BRCT domain heterodimer for the first time by mutating tyrosine 574 in mouse XRCC1 (corresponding to tyrosine 576 in human XRCC1) that is subject to a polymorphism that previously had been demonstrated not to perturb the function of the protein. Introduction of a non-conservative Y574R mutation blocked crystallization of the XRCC1 BRCT domain homodimer, allowing crystallization of the heterodimer. Based on the initial structure obtained, it appeared that heterodimer formation could be enhanced by inclusion of a longer construct of the XRCC1 that included residues formally part of the linker domain preceding the C-terminal BRCT domain. Inclusion of these residues resulted in enhanced affinity for the Ligase3alpha BRCT domain, and the additional interactions were also characterized structurally. Thus, the enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity of the interaction. Project 2. We have previously demonstrated that the N-terminal, DNA polymerase beta-interactive domain of XRCC1 can adopt either of two structures, one of which contains a disulfide bond. Disulfide switches are increasingly recognized as a component of the cellular response to oxidative stress, and it is therefore likely that this alternate conformation plays a role in the regulation of the XRCC1 activity under such conditions. We have recently developed a more convenient NMR approach to monitoring the reduced/oxidized ratio of the protein, which allows us to further evaluate how various experimental conditions influence this ratio. It is anticipated that once we have identified conditions that stabilize the oxidized form of the protein, we can move forward toward the identification of additional binding partners that are selective for the oxidized form of the domain. Thus, as noted previously, nearly all of the structural changes that accompany dislulfide bond formation are located on the face of the molecule that is opposite to the face that interacts with DNA pol beta. Project 3. Recruitment of XRCC1 and Lig3 to single strand breaks in chromatin requires the activation of poly(ADP-ribose) polymerase-1 (PARP1) activity; PARP1 is one of the first proteins localized to foci of DNA damage. Upon activation by encountering nicked DNA, the PARP1-mediated trans-poly(ADP-ribosyl)ation of DNA binding proteins occurs, facilitating access and accumulation of DNA repair factors. PARP1 also auto-(ADP-ribosyl)ates its central BRCT-containing domain forming part of an interaction site for the DNA repair scaffolding protein X-ray cross complementing group 1 protein (XRCC1). The co-localization of XRCC1, as well as bound DNA repair factors, to sites of DNA damage is important for cell survival and genomic integrity. During the past review period, we determined the solution structure of the PARP1 BRCT domain, and also characterized the behavior and interactions of the isolated domain. The PARP1 BRCT domain has the globular a/b fold characteristic of BRCT domains and has a thermal melting transition of 43.0C. In contrast to a previous characterization of this domain, we found that it is monomeric in solution using both gel-filtration chromatography and small-angle X-ray scattering. Also in contrast with some earlier reports, we determined that the first BRCT domain of XRCC1 does not interact significantly with the PARP1 BRCT domain in the absence of ADP-ribosylation. Moreover, none of the interactions with other longer PARP1 constructs which previously had been demonstrated in a pull-down assay of mammalian cell extracts were detected. Our data indicating no significant interaction between the BRCT domains of PARP1 and XRCC1 likely results from the absence of poly(ADP-ribose) in one or both binding partners, and further implicates a poly(ADP-ribose)-dependent mechanism for localization of XRCC1 to sites of DNA damage. Project 4. It has recently been demonstrated that misincorporation 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(GCG)rCd(TTAAGCGC), contains two symmetrically positioned cytidine residues paired with deoxyguanosine residues. The absence of an observable H1&#8722;H2 scalar coupling interaction indicates a C3&#8242;-endo conformation for the ribose. Longer-range structural perturbations resulting from the presence of the ribonucleotide are limited to the adjacent and transhelical nucleotides, while the global B-form DNA structure is maintained. Because crystallographic studies have indicated that isolated ribonucleotides promote global B &#8594; A transitions, we also performed molecular modeling analyses to evaluate the structural consequences of higher ribonucleotide substitution levels. Increasing the ribonucleotide content increased the minor groove width toward values more similar to that of A-DNA, but even 50% ribonucleotide substitution did not fully convert the B-DNA to A-DNA. Comparing our structure with the structure of an RNase H2-bound DNA supports the conclusion that, as with other DNA&#8722;protein complexes, the DNA conformation is strongly influenced by the interaction with the protein.