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      End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining

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          Abstract

          DNA polymerase μ is a member of the mammalian pol X family and reduces deletion during chromosome break repair by nonhomologous end joining (NHEJ). This biological role is linked to pol μ's ability to promote NHEJ of ends with noncomplementary 3′ overhangs, but questions remain regarding how it performs this role. We show here that synthesis by pol μ in this context is often rapid and, despite the absence of primer/template base-pairing, instructed by template. However, pol μ is both much less active and more prone to possible template independence in some contexts, including ends with overhangs longer than two nucleotides. Reduced activity on longer overhangs implies pol μ is less able to synthesize across longer gaps, arguing pol μ must bridge both sides of gaps between noncomplementary ends to be effective in NHEJ. Consistent with this argument, a pol μ mutant defective specifically on gapped substrates is also less active during NHEJ of noncomplementary ends both in vitro and in cells. Taken together, pol μ activity during NHEJ of noncomplementary ends can thus be primarily linked to pol μ's ability to work together with core NHEJ factors to bridge DNA ends and perform a template-dependent gap fill-in reaction.

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          Most cited references 28

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          The complexity of DNA damage: relevance to biological consequences.

          Ionizing radiation causes both singly and multiply damaged sites in DNA when the range of radical migration is limited by the presence of hydroxyl radical scavengers (e.g. within cells). Multiply damaged sites are considered to be more biologically relevant because of the challenges they present to cellular repair mechanisms. These sites occur in the form of DNA double-strand breaks (dsb) but also as other multiple damages that can be converted to dsb during attempted repair. The presence of a dsb can lead to loss of base sequence information and/or can permit the two ends of a break to separate and rejoin with the wrong partner. (Multiply damaged sites may also be the biologically relevant type of damage caused by other agents, such as UVA, B and/or C light, and some antitumour antibiotics.) The quantitative data available from radiation studies of DNA are shown to support the proposed mechanisms for the production of complex damage in cellular DNA, i.e. via scavengable and non-scavengable mechanisms. The yields of complex damages can in turn be used to support the conclusion that cellular mutations are a consequence of the presence of these damages within a gene. Literature data are used to support these statements and to develop overall mechanisms connecting the production of primary species to the production of biologically relevant damages. The consequences of the LET of the radiation on multiplicity of damage are discussed and suggestions made for the cause of the decrease of the oxygen enhancement ratio as the LET increases.
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            A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining.

            Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear. We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ. However, only pol mu promotes accuracy during immunoglobulin kappa recombination. This distinctive in vivo role correlates with the TdT-like ability of pol mu, but not pol lambda, to act when primer termini lack complementary bases in the template strand. However, unlike TdT, synthesis by pol mu in this context is primarily instructed by a template from another DNA molecule. This apparent gradient of template dependence is largely attributable to a small structural element that is present but different in all three polymerases.
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              A biochemically defined system for mammalian nonhomologous DNA end joining.

              Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                May 2008
                8 April 2008
                8 April 2008
                : 36
                : 9
                : 3085-3094
                Affiliations
                1Department of Biochemistry and Biophysics, 2Lineberger Comprehensive Cancer Center and 3Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, NC 27599, USA
                Author notes
                *To whom correspondence should be addressed. +1 919 966 9809+1 919 966 3015 dale_ramsden@ 123456med.unc.edu
                Article
                gkn164
                10.1093/nar/gkn164
                2396419
                18397950
                © 2008 The Author(s)

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Nucleic Acid Enzymes

                Genetics

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