Solution Structure of Polymerase μ's BRCT Domain Reveals an Element Essential for Its Role in Nonhomologous End Joining †

Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, North Carolina, United States
Biochemistry (Impact Factor: 3.02). 10/2007; 46(43):12100-10. DOI: 10.1021/bi7007728
Source: PubMed


The solution structure and dynamics of the BRCT domain from human DNA polymerase mu, implicated in repair of chromosome breaks by nonhomologous end joining (NHEJ), has been determined using NMR methods. BRCT domains are typically involved in protein-protein interactions between factors required for the cellular response to DNA damage. The pol mu BRCT domain is atypical in that, unlike other reported BRCT structures, the pol mu BRCT is neither part of a tandem grouping, nor does it appear to form stable homodimers. Although the sequence of the pol mu BRCT domain has some unique characteristics, particularly the presence of >10% proline residues, it forms the characteristic alphabetaalpha sandwich, in which three alpha helices are arrayed around a central four-stranded beta-sheet. The structure of helix alpha1 is characterized by two solvent-exposed hydrophobic residues, F46 and L50, suggesting that this element may play a role in mediating interactions of pol mu with other proteins. Consistent with this argument, mutation of these residues, as well as the proximal, conserved residue R43, specifically blocked the ability of pol mu to efficiently work together with NHEJ factors Ku and XRCC4-ligase IV to join noncomplementary ends together in vitro. The structural, dynamic, and biochemical evidence reported here identifies a functional surface in the pol mu BRCT domain critical for promoting assembly and activity of the NHEJ machinery. Further, the similarity between the interaction regions of the BRCT domains of pol mu and TdT support the conclusion that they participate in NHEJ as alternate polymerases.

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Available from: Geoffrey Andrew Mueller, Mar 17, 2015
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    • ", or point-mutagenesis of keyresidues [33] [36], block the formation of complexes between the polymerase, Ku and XRCC4/ LigaseIV at DNA ends. "
    New Research Directions in DNA Repair, Edited by Clark Chen, 05/2013: chapter Evolving DNA Repair Polymerases: From Double—Strand Break Repair to Base Excision Repair and VDJ Recombination; InTech., ISBN: 978-953-51-1114-6
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    • "Nevertheless, all three interact specifically with Ku, XRCC4-ligase IV, and DNA ends, and a-helix1 is critical to this interaction in all three examples (Fig. 3). An arginine (R43) and exposed hydrophobic residues (F46 and L50) in this helix are conserved through vertebrate Pol l and TdT [DeRose et al., 2007], and are important for both interaction of Pol l with Ku and XRCC4-ligase IV at DNA ends as well as Pol l activity in NHEJ. There are analogous residues in Pol k for the first two positions (R57 and L60), and these residues are also important for NHEJ interactions [Mueller et al., 2008], but R57 in Pol k is displaced relative to R43 in Pol l. "
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    ABSTRACT: Chromosome breaks, often with damaged or missing DNA flanking the break site, are an important threat to genome stability. They are repaired in vertebrates primarily by nonhomologous end joining (NHEJ). NHEJ is unique among the major DNA repair pathways in that a continuous template cannot be used by DNA polymerases to instruct replacement of damaged or lost DNA. Nevertheless, at least 3 out of the 17 mammalian DNA polymerases are specifically employed by NHEJ. Biochemical and structural studies are further revealing how each of the polymerases employed by NHEJ possesses distinct and sophisticated means to overcome the barriers this pathway presents to polymerase activity. Still unclear, though, is how the resulting network of overlapping and nonoverlapping polymerase activities contributes to repair in cells. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.
    Environmental and Molecular Mutagenesis 12/2012; 53(9). DOI:10.1002/em.21725 · 2.63 Impact Factor
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    • "Interestingly, sequence comparisons show that the BRCT of Polµ is most similar to TdT, with 39% sequence identity that includes the residues important for complex formation (40). This high level of sequence conservation is maintained at a 3D-structural level, in the BRCT domains of Polµ (PDB ID: 2DUN) and TdT (PDB ID: 2COE), as well as in their electrostatic surfaces, containing both a positively charged ridge on one face of the protein, and large negatively charged regions on the opposite faces. "
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    ABSTRACT: Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys(249), Arg(253) and Arg(416)) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.
    Nucleic Acids Research 10/2012; 40(22). DOI:10.1093/nar/gks896 · 9.11 Impact Factor
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