DNA joint dependence of Pol X family polymerase action in nonhomologous end joining

University of Michigan, Ann Arbor, Michigan, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2005; 280(32):29030-7. DOI: 10.1074/jbc.M505277200
Source: PubMed


DNA double strand breaks (DSBs) can be rejoined directly by the nonhomologous end-joining (NHEJ) pathway of repair. Nucleases and polymerases are required to promote accurate NHEJ when the terminal bases of the DSB are damaged. The same enzymes also participate in imprecise rejoining and joining of incompatible ends, important mutagenic events. Previous work has shown that the Pol X family polymerase Pol4 is required for some but not all NHEJ events that require gap filling in Saccharomyces cerevisiae. Here, we systematically analyzed DSB end configurations and found that gaps on both strands and overhang polarity are the principal factors that determine whether a joint requires Pol4. DSBs with 3'-overhangs and a gap on each strand strongly depended on Pol4 for repair, DSBs with 5'-overhangs of the same sequence did not. Pol4 was not required when 3'-overhangs contained a gap on only one strand, however. Pol4 was equally required at 3'-overhangs of all lengths within the NHEJ-dependent range but was dispensable outside of this range, indicating that Pol4 is specific to NHEJ. Loss of Pol4 did not affect the rejoining of DSBs that utilized a recessed microhomology or DSBs bearing 5'-hydroxyls but no gap. Finally, mammalian Pol X polymerases were able to differentially complement a pol4 mutation depending on the joint structure, demonstrating that these polymerases can participate in yeast NHEJ but with distinct properties.

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    • "In yeast, the recruited Mre11/Rad50/Xrs2 complex (MRX), required for NHEJ, has been also proposed to bind and connect two DNA molecules [1]. Those termini are further processed by nucleases (Artemis, Fen1), phosphatases and kinases generating gapped intermediates filled by a DNA polymerase belonging to family X (λ, µ and yeast Pol IV) [2], [3], [12]–[14]. Finally, the complex Ligase IV/XRCC4/XLF (Dnl4/Lif1/Nej1 in yeast) joins the ends together [1], [15]. "
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    ABSTRACT: It is widely accepted that repair of double-strand breaks in bacteria that either sporulate or that undergo extended periods of stationary phase relies not only on homologous recombination but also on a minimal nonhomologous end joining (NHEJ) system consisting of a dedicated multifunctional ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku. Bacillus subtilis is one of the bacterial members with a NHEJ system that contributes to genome stability during the stationary phase and germination of spores, having been characterized exclusively in vivo. Here, the in vitro analysis of the functional properties of the purified B. subtilis LigD (BsuLigD) and Ku (BsuKu) proteins is presented. The results show that the essential biochemical signatures exhibited by BsuLigD agree with its proposed function in NHEJ: i) inherent polymerization activity showing preferential insertion of NMPs, ii) specific recognition of the phosphate group at the downstream 5' end, iii) intrinsic ligase activity, iv) ability to promote realignments of the template and primer strands during elongation of mispaired 3' ends, and v) it is recruited to DNA by BsuKu that stimulates the inherent polymerization and ligase activities of the enzyme allowing it to deal with and to hold different and unstable DNA realignments.
    PLoS ONE 05/2013; 8(5):e64232. DOI:10.1371/journal.pone.0064232 · 3.23 Impact Factor
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    • "Like Polµ, SpPol4 can incorporate an NTP into a primer DNA. Also, mammalian family X polymerases have been shown to differentially complement a Δpol4 mutation in Saccharomyces cerevisiae, depending on the joint structure, demonstrating that these polymerases can participate in yeast NHEJ but with distinct specificities (31). In the light of all this evidence, we decided to use S. pombe as a model organism, as it provides us with a simplified system to test the usage of ribonucleotide substrates by SpPol4, Pol µ and Polλ (over-expressed in this system) with a more physiological approach: using whole cell extracts instead of purified recombinant proteins. "
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    ABSTRACT: Human DNA polymerase mu (Polmu), a family X member involved in DNA repair, has both template-directed and terminal transferase (template-independent) activities. In addition to their ability to incorporate untemplated nucleotides, another similarity between Polmicro and terminal deoxynucleotidyl transferase (TdT) is their promiscuity in using ribonucleotides (NTPs), whose physiological significance is presently unknown. As shown here, Polmicro can use NTPs instead of deoxynucleotides (dNTPs) during non-homologous end joining (NHEJ) of non-complementary ends, a Polmicro-specific task. Moreover, a physiological concentration of Mn(2+) ions did benefit Polmicro-mediated NHEJ by improving the efficiency and accuracy of nucleotide insertion. Analysis of different mutations in the 'steric gate' of the active site indicated that Polmicro is taking advantage of an open active site, valid for selecting alternative activating metal ions and nucleotides as substrates. This versatility would allow ad hoc selection of the most appropriate nucleotide/metal ion combination for individual NHEJ events to gain efficiency without a cost in terms of fidelity, thus widening the spectrum of available solutions to position a discontinuous template strand in proper register for connection.
    Nucleic Acids Research 12/2012; 41(4). DOI:10.1093/nar/gks1444 · 9.11 Impact Factor
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    • "Pol b, consequently, does not stably associate with NHEJ core factors at ends, nor does it effectively promote NHEJ in many contexts in vitro [Mahajan et al., 2002; Ma et al., 2004; Nick McElhinny et al., 2005]. Nevertheless, Pol b is able to partly compensate for Pol4 deficiency during NHEJ when overexpressed in yeast [Daley et al., 2005] and Pol b-deficient mouse embryonic fibroblasts (MEFs) are moderately sensitive to ionizing radiation [Horton et al., 2008]. Unlike other vertebrate Pol X members, Pol b deficiency results in embryonic lethality in mice [Gu et al., 1994; Bertocci et al., 2006]. "
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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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