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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Changing macromolecular conformations and complexes are critical features of cellular networks, typified by DNA damage response pathways that are essential to life. These fluctuations enhance specificity of macromolecular recognition and catalysis, and enable an integrated functioning of pathway components, ensuring efficiency while reducing off pathway reactions. Such dynamic complexes challenge classical detailed structural analyses, so there characterizations demand combining methods that provide detail with those that inform dynamics in solution. Small angle xray scattering, electron microscopy, hydrogen-deuterium-exchange and computation are complementing detailed structures from crystallography and NMR to provide comprehensive models for DNA damage searching, specificity, signaling and repair. Here, we review new approaches and results on DNA damage responses that advance structural biology in the fourth dimension, connecting proteins to pathways.

primosome disassembly, thereby increasing primase processivity. In contrast, priming in bacteriophage T7 involves discrete pausing of the primosome, and in Escherichia coli it appears to be associated primarily with dissociation of the primase from the helicase. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.