Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases DNA nick-joining and intermolecular DNA ligation. Yet, the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small angle x-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) form a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) -OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick-sensing and the transition from nick-sensing by the ZnF to nick-joining by the catalytic core. The collective results support a "jackknife model" whereby the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.DNA ligase III is a vertebrate-specific protein functioning in DNA replication and repair pathways, including nucleotide excision repair, base excision repair, and single-strand break repair, plus mitochondrial replication and repair (1). DNA ligase III is furthermore † This work was supported in part by grants from the National Institutes of Health (NIH) including (5R01 GM052504; TE), and The Supporting Information AvailableWe provide additional figures and experimental procedures in the supporting information. This material is available free of charge via the Internet at: http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 July 27. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript implicated in the repair of DNA double-strand breaks when nonhomologous end joining (NHEJ) activity is compromised (2). Upregulated ligase III expression in chronic myeloid leukemia cells, with concomitant decreases in the expression of the NHEJ proteins DNA ligase IV and Artemis, may promote cell survival and disease progression, raising the possibility of selectively inhibiting ligase III as a cancer treatment (3). Besides repairing nuclear DNA, ligase III is the only mitochondrial DNA ligase where it functions in DNA repair and replication.Three DNA ligase III isoforms are generated by alternative mRNA splicing and translation initiation, and expression of one or more of these is essential for the viability of mammalian cells and animals (4). The LigIIIα isoform interacts with XRCC1 through a C-terminal BRCA1-related C-terminal (BRCT) domain, and this protein complex functions in a variety of DNA repair pathways, most prominently in the repair of DNA single-strand breaks (5,6). LigIIIβ lacks the C-terminal BRCT domain (6,7), and is expressed only in the male germ line where it presumably ...

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Background:The DNA repair scaffold XRCC1 binds to poly(ADP-ribose)ylated PARP1 at damaged chromatin. Results: XRCC1 preferentially binds to poly(ADP-ribose) chains longer than 7 ADP-ribose units in length. Conclusion: We identify specific determinants of XRCC1-PARP1 complex assembly, and disassembly by PARG. Significance: Our TR-FRET assay is useful for investigating turnover of posttranslational modifications and for identifying inhibitors by high-throughput screening.

Background: Proper regulation of DSB resection is key to genome maintenance. Results: 14-3-3s bind to Exo1 and restrain its damage association and resection activity by counteracting the function of PCNA. Conclusion: Exo1 activity is controlled by both positive and negative regulators to ensure a proper level of DNA end resection. Significance: Our data reveal a key mechanism that controls the DNA end resection process.

The XRCC1–DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT–BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS–MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT–BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.