FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2053016947> ?p ?o ?g. }

• W2053016947 endingPage "1023" @default.
• W2053016947 startingPage "1010" @default.
• W2053016947 abstract "Article8 February 2007free access XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps Jiafeng Gu Jiafeng Gu Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA Department of Biological Sciences, Los Angeles, CA, USA Search for more papers by this author Haihui Lu Haihui Lu Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA Search for more papers by this author Brigette Tippin Brigette Tippin Department of Biological Sciences, Los Angeles, CA, USAPresent address: Division of Medical Genetics, Department of Pediatrics, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, 1124 W Carson Street, Torrance, CA 90502, USA Search for more papers by this author Noriko Shimazaki Noriko Shimazaki Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA Search for more papers by this author Myron F Goodman Myron F Goodman Department of Biological Sciences, Los Angeles, CA, USA Search for more papers by this author Michael R Lieber Corresponding Author Michael R Lieber Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA Department of Biological Sciences, Los Angeles, CA, USA Search for more papers by this author Jiafeng Gu Jiafeng Gu Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA Department of Biological Sciences, Los Angeles, CA, USA Search for more papers by this author Haihui Lu Haihui Lu Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA Search for more papers by this author Brigette Tippin Brigette Tippin Department of Biological Sciences, Los Angeles, CA, USAPresent address: Division of Medical Genetics, Department of Pediatrics, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, 1124 W Carson Street, Torrance, CA 90502, USA Search for more papers by this author Noriko Shimazaki Noriko Shimazaki Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA Search for more papers by this author Myron F Goodman Myron F Goodman Department of Biological Sciences, Los Angeles, CA, USA Search for more papers by this author Michael R Lieber Corresponding Author Michael R Lieber Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA Department of Biological Sciences, Los Angeles, CA, USA Search for more papers by this author Author Information Jiafeng Gu1,2, Haihui Lu1, Brigette Tippin2, Noriko Shimazaki1, Myron F Goodman2 and Michael R Lieber 1,2 1Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA 2Department of Biological Sciences, Los Angeles, CA, USA *Corresponding author. Norris Cancer Ctr., Rm. 5428, University of Southern California, 1441 Eastlake Ave., MC 9176, Los Angeles, CA 90033, USA. Tel.: +1 323 865 0568; Fax: +1 323 865 3019; E-mail: [email protected] The EMBO Journal (2007)26:1010-1023https://doi.org/10.1038/sj.emboj.7601559 Correction(s) for this article XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps25 July 2007 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the nonhomologous DNA end joining pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3′ overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1–4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nt. Ku can stimulate the joining, but is not essential when there is some terminal annealing. Polymerase mu can add nucleotides in a template-independent manner under physiological conditions; and the subset of ends that thereby gain some terminal microhomology can then be ligated. Hence, annealing at sites of microhomology is very important, but the flexibility of the ligase complex is paramount in nonhomologous DNA end joining. These observations provide an explanation for several in vivo observations that were difficult to understand previously. Introduction Double-strand DNA breaks (DSBs) in mammalian cells are repaired predominantly by either nonhomologous DNA end joining (NHEJ) or homologous recombination (Ma et al, 2005a; Friedberg et al, 2006). NHEJ of DSBs is thought to begin with the binding of the heterodimer Ku to the double-stranded DNA (dsDNA) ends. Like many repair processes, NHEJ utilizes nucleases, polymerases, and ligases; and Ku functions as a ‘toolbelt’ protein by recruiting each of these enzymatic components (Ma et al, 2004). The ligase complex is XRCC4:DNA ligase IV in all eukaryotes (Grawunder et al, 1997; Schar et al, 1997; Teo and Jackson, 1997; Wilson et al, 1997; Tomkinson et al, 2006). XLF or Cernunnos in mammalian cells is a homologue of Saccharomyces cerevisiae NEJ1 and forms a complex with XRCC4:DNA ligase IV, the functional consequences of which are yet to be defined (Ahnesorg et al, 2006; Buck et al, 2006; Callebaut et al, 2006). Ku improves the binding of the XRCC4:DNA ligase IV complex to DNA ends (Chen et al, 2000; NickMcElhinny et al, 2000). Ku also improves the on-rate and slows the off-rate of DNA-PKcs from DNA ends (Gottlieb and Jackson, 1993; West et al, 1998). A significant fraction of Artemis exists in the cell in complex with DNA-PKcs, and becomes an endonuclease after it is phosphorylated by DNA-PKcs (Ma et al, 2002). There are 11 DNA-PKcs phosphorylation sites in the C-terminal half of Artemis (Ma et al, 2005b). Phosphorylation of these sites by DNA-PKcs causes a conformational change in Artemis, allowing it to trim 5′ overhangs to a blunt conformation and trim long 3′ overhangs to much shorter ones, typically about 4 nt in length (Ma et al, 2005b). DNA polymerases (pol) are thought to be needed for any NHEJ events that require fill-in of gaps or extension of the 3′ end at 5′ overhangs (Ma et al, 2005a). POL4 is responsible for a substantial fraction of the fill-in synthesis of gaps in NHEJ events in S. cerevisiae (Wilson and Lieber, 1999). POL4 is a member of the Pol X family and is most homologous to pol mu and pol lambda in mammalian cells (Tseng and Tomkinson, 2002). The only other two known mammalian proteins in the Pol X family are pol beta and the lymphoid-specific enzyme, terminal deoxynucleotidyl transferase (TdT). Ku recruits pol mu and pol lambda to DNA ends via their BRCT domains (Ma et al, 2004). One group has proposed that pol mu can polymerize across a discontinuous template strand, whereas pol lambda was not reported to do so (NickMcElhinny et al, 2005). This ‘jumping’ from one DNA end to another was proposed as a basis for why pol mu deficiency results in an altered V(D)J recombination phenotype, where joining of two DNA ends with incompatible 3′ overhangs is suspected to be common (Schlissel, 1998). However, both the pol lambda null mice and the pol mu null mice turn out to have phenotypes in V(D)J recombination (Bertocci et al, 2003, 2006), raising questions about models based on different abilities of pol mu and pol lambda to synthesize across discontinuous templates. A fundamental question in NHEJ is the relative contribution of protein factors in configuring the ends for ligation and the role of the intrinsic terminal DNA sequence (Daley et al, 2005b). Here we find that XRCC4:DNA ligase IV plus Ku can ligate several fully incompatible DNA end configurations that do not share even 1 bp of terminal microhomology. Moreover, XRCC4:DNA ligase IV can ligate across short gaps. Terminal annealing of 1–4 bp can obviate the need for Ku, although it is often still stimulatory. When confronted with incompatible DNA ends, pol mu can add nucleotides in a template-independent manner, thereby creating random microhomology in a subset of DNA ends. Pol lambda has only a marginal template-independent polymerase activity. Hence, the template-independent activity of pol mu and the remarkable flexibility of the ligase complex permit a wide range of incompatible DNA ends to be joined via NHEJ. Results Pol mu has template-independent polymerase activity under physiological conditions and has a preference for pyrimidine addition Previous work suggested that human pol mu has template-independent polymerase activity, but nearly all of this work was carried out using Mn2+ as the divalent cation (Dominguez et al, 2000; Garcia-Diaz et al, 2000; Ramadan et al, 2003, 2004; Juarez et al, 2006). Data for Mg2+ conditions were limited to a single-stranded DNA substrate (Dominguez et al, 2000) or to substrates with long (10–20 nt) 5′ overhangs (Covo et al, 2004). We were interested in testing whether pol mu has template-independent polymerase activity under physiological conditions (Mg2+ present) at dsDNA ends of the type subject to NHEJ. Our substrates for these studies consist of 73 bp dsDNA with an additional 2 nt overhang at each 3′ end (Figure 1A). We used substrates where both ends have an –AG overhang or where both ends have a –TC overhang. The DNA substrates are 5′-radiolabeled with 32P at one end, but the other DNA end has a 5′ OH. We added pol mu to these substrates in free solution, and in selected reactions we also added Ku and XRCC4:DNA ligase IV (Figure 1B). After incubation to permit addition of nucleotides by pol mu, we analyzed the products using denaturing PAGE. Figure 1.Template-independent polymerase activity of pol mu on substrates in free solution. (A) Two 73 bp substrates with 3′ overhangs were used to test for pol mu template-independent polymerase activity in free solution. An asterisk indicates the position of the radioisotope label. (B, C) In each reaction, 20 nM substrate was incubated with the protein(s) indicated above each lane in a 10 μl reaction for 1 h at 37°C. After incubation, reactions were deproteinized and analyzed using 11% denaturing PAGE. Protein concentrations: Ku, 25 nM; X4-LIV, 50 nM; pol mu, 25 nM, where X4-LIV refers to XRCC4:DNA ligase IV. The specified dNTP was added to 100 μM. ‘dN’ means that all the four dNTPs (100 μM each) were included. No ATP was added, unless specified. Template-independent polymerase synthesis results in extension of the radiolabeled strand, and hence, the more slowly moving species located above the substrate band. Download figure Download PowerPoint We found that pol mu alone had little or no template-independent activity under these conditions (Figure 1B, lanes 2 and 12). But when Ku and XRCC4:DNA ligase IV were present, we noted significant mononucleotide addition to the 3′ overhanging ends (Figure 1B, lanes 7–10 and 17–20). On the basis of single nucleotide iterations, pol mu appears to carry out distributive synthesis on both substrates here. The presence of both Ku and XRCC4:DNA ligase IV were important to achieve maximal pol mu nucleotide addition (Figure 1C, lanes 9 and 10 versus 1–8; also Figure 1B). This is consistent with the fact that Ku can bind to the BRCT domain of pol mu (Ma et al, 2004), and that XRCC4:DNA ligase IV may provide additional stability for the Ku:pol mu interaction (Mahajan et al, 2002). Interestingly, the two types of DNA ends do not give identical results. Total nucleotide addition to the –AG end is more efficient than to the –TC end. This may reflect better initial binding of the pol mu to this terminal sequence. T is not only the obvious preferred nucleotide for the –AG end (Figure 1B, lane 10), but is seen on longer exposure to be the nucleotide associated with the longest additions at the –TC end also (Supplementary Figure 1, lane 20). However, it is important to note that all four dNTPs could be added to each of the two types of overhangs (Figure 1B, lanes 7–10 and 17–20). Otherwise, one would expect C and T addition only to the –AG end and G and A to the –TC end, and this is clearly not the case. Therefore, pol mu has template-independent activity under physiological conditions. Polymerization by pol mu at free DNA ends is template-independent and not due to use of another DNA end as the source of a template strand Pol mu has also well-documented template-dependent activity (Dominguez et al, 2000; Supplementary Figure 4). To definitively rule out any possibility of template-dependent addition by pol mu, we immobilized the DNA at one end to streptavidin–agarose beads using biotin. The DNA was used at a level 100- to 1000-fold below the capacity of the beads to ensure that the DNA ends could not contact one another. Moreover, we showed that the DNA ends of the bead-bound substrates could no longer ligate, verifying the highly dispersed distribution of the DNA substrate on the beads (Supplementary Figure 2B, which is a darker exposure of Figure 2). Polymerase synthesis studies were conducted similar to those described above, using the same types of DNA ends (Figure 2). We still observe template-independent addition that cannot be explained by using another DNA end as a template (NickMcElhinny et al, 2005). Specifically, pol mu could add any of the four nucleotides to the –AG or to the –TC ends (Figure 2B, lanes 2–5 and 9–12). Not surprisingly, the overall efficiency of the reaction on the bead was lower. Therefore, to observe products, we increased the amount of polymerase used. Yet we still observe that any of the four nucleotides can be added. Therefore, pol mu can add nucleotides in a template-independent manner under physiological conditions to 3′ overhangs. Figure 2.Template-independent synthesis by pol mu on immobilized DNA substrates distributed at low density on agarose beads. (A) Streptavidin agarose beads were used to immobilize two 73 bp DNA substrates with 3′ overhangs. ‘B’ designates the 3′-biotin group of the substrate. An asterisk indicates the position of the radioisotope label. (B) In each reaction, 20 nM substrate was incubated with the protein(s) indicated above each lane in a 20 μl reaction for 1 h at 37°C. After incubation, reactions were heated at 100°C for 5 min to disrupt the biotin–streptavidin interaction, and then deproteinized and analyzed using 11% denaturing PAGE. Protein concentrations: Ku, 50 nM; X4-LIV, 100 nM; pol mu, 1.25 μM; dNTP, 5 mM. No ATP was added. Download figure Download PowerPoint There are some interesting additional points to note. First, C and T are the preferred nucleotide additions at both the –AG and the –TC ends (Supplementary Figure 2, lanes 3 and 5 for the –AG ends and lanes 10 and 12 for the –TC ends). It is clear that over 20 C and T nts can be added to the free –AG ends. For the –TC end, over 20 C nts can be added, and addition of T also clearly occurs, but not as efficiently as C. Hence, pyrimidines seem to be added better than purines, regardless of the sequence of the end. The difference between the number of nucleotides added at –AG and –TC ends may relate to how the polymerase contacts the DNA at the double- to single-strand transition region. Second, nucleotide addition by pol mu alone without Ku and XRCC4:DNA ligase IV is substantial for the immobilized substrates (Figure 2, lanes 6 and 13). This may mean that immobilization of the DNA makes it easier for pol mu to add nucleotides in a template-independent manner, even without the stabilizing influence of Ku and XRCC4:DNA ligase IV. We did the same type of study for template-independent addition to the free end of bead-bound DNA substrates, but we replaced the dNTPs with the corresponding ribonucleotides (NTPs). Previous work had shown that pol mu can perform fill-in synthesis using NTPs (NickMcElhinny et al, 2005). However, template-independent synthesis with NTPs had not been reported. Not surprisingly, like dNTPs, the corresponding NTPs can be added in a template-independent manner (Supplementary Figure 3). In summary, these immobilized substrate experiments demonstrate that pol mu can carry out robust template-independent synthesis under physiological conditions, and the level of this activity suggests that this is the dominant mode of addition at a free DNA end with a 3′ overhang. We also studied human pol lambda, which previously was shown to have template-independent activity, but only in Mn2+ solutions (Ramadan et al, 2003). At equivalent molar amounts, pol lambda has a similar or even slightly higher template-dependent activity than pol mu (Supplementary Figure 4). However, when tested for template-independent activity, we observed only a very marginal level, regardless of addition of Ku or XRCC4:DNA ligase IV, and regardless of whether the DNA substrate is free in solution or immobilized on beads (data not shown; see below). The template-independent synthesis by pol mu provides the microhomology for end ligaton by Ku, XRCC4, and DNA ligase IV Having gained a better understanding of the polymerization properties of pol mu, we were interested in understanding the role of pol mu in the ligation of the same types of DNA ends described above (Figure 3A). For any given ligation reaction, duplex DNA substrates with 2 nt overhangs were used. One 5′ end was radiolabeled with 32P, and the other 5′ end was not ligatable because it had a 5′ OH. To carry out the ligation reactions, we added Ku, XRCC4:DNA ligase IV, dNTPs, and either pol mu or pol lambda. We incubated reactions for 30 min, deproteinized, and analyzed with denaturing PAGE. Figure 3.Pol mu template-independent polymerase activity provides terminal microhomology for ligation by XRCC4:DNA ligase IV. (A) The same substrate as in Figure 1A (left side) was tested for ligation. Two alternative joining pathways are proposed below the substrate. From the ligation patterns in (B) and Supplementary Figure 5, we know that the first pathway is favored. An asterisk indicates the position of the radioisotope label. (B) In each reaction, 20 nM substrate was incubated with the protein(s) indicated above each lane in a 10 μl reaction for 30 min at 37°C. After incubation, reactions were deproteinized and analyzed using 8% denaturing PAGE. Protein concentrations: Ku, 25 nM; X4-LIV, 50 nM; pol mu or lambda, 25 nM. dNTP (100 μM) was added to reactions, where indicated. ‘dN’ means all the four dNTPs (100 μM each) were included. ATP (100 μM) was also added in indicated reactions. ‘M’ indicates 50 bp DNA ladder. The dimer ligation product that results from the joining of two substrate molecules is labeled. Joining of more than two substrates results in trimer and higher-order species labeled as multimers. (C) Dimer products from the selected lanes were cut out of the gel, extracted, and then PCR amplified, TA-cloned, and sequenced. The junction sequences for the ligatable strand were provided. For lane 3, sequencing information was collected and combined from three individual reactions. For lane 8, two bands are apparent in the dimer product, but the longer product was not among the four molecules sequenced. Download figure Download PowerPoint Even when Ku, XRCC4:DNA ligase IV, and pol mu are all present, we find that the ligation depends on the presence of specific dNTPs that could potentially provide 1 or 2 bp of complementarity (Figure 3B, lanes 6, 8, and 9 versus 4, 5 and 7). To confirm this, we cut out the dimer species from the gel, PCR amplified across the ligation junction, cloned into a TA cloning vector, transformed bacteria, isolated the plasmid, and sequenced the individual ligation junctions (Figure 3C). For reactions in which only dTTP is present, the junctions only contain a T addition, which provides 1 bp of terminal homology. The resulting single A:T bp is adequate to support the ligation. For reactions that contain all four dNTPs (Figure 3B, lane 3), sequencing of the dimer product shows junctions with a spectrum of additions, but predominantly a single T (Figure 3C, upper). As expected based on template-independent synthesis at a DNA end, the number of junctions with CT additions, which would provide 2 bp of terminal homology, was considerably smaller. A few junctions illustrated additional template-independent addition which ended in CT, providing complementarity, but which showed addition of other nucleotides 5′ to the CT. Most of the added nucleotides were pyrimidines, illustrating the pyrimidine preference noted earlier. Hence, in the presence of Ku and XRCC4:DNA ligase IV, pol mu can support template-independent addition at DNA ends, and the resulting subset of ends that acquire complementarity can now be ligated. Substrates with a –TC overhang can be ligated when dATP or dGTP are provided (Supplementary Figure 1B, lanes 17 and 19). When the dimer ligation products are sequenced, the expected A or G nts are present, consistent with the terminal microhomology for joining being provided by template-independent addition. The above results indicate that 1 bp of annealing between two DNA ends is sufficient to support ligation, even when the other strand remains in an unligatable configuration. Given this, we reasoned that if we provide that one specific base pair of terminal microhomology in the starting substrate, then the ligation should proceed even without any polymerase present. We found that, indeed, this is the case (Figure 4B, lane 4 on both panels). A substantial amount of dimer and higher ligation multimers can be formed when the one DNA end overhang is –AGT and is ligated to a DNA end with an incompatible 3′ overhang (Figure 4B, right panel, lane 3). The bottom strand in this case would remain unligated for three reasons: because there is no 5′ phosphate at the junction; there is a gap on the bottom strand; and the right bottom strand has a 1 nt 3′ flap. Figure 4.One base pair of terminal microhomology is sufficient for direct ligation by XRCC4:DNA ligase IV. (A) Two substrates with only 1 bp of terminal microhomology for ligation were designed, based on the Figure 3A substrate, to test the direct ligation by XRCC4:DNA ligase IV. Two alternative joining products are proposed below each substrate. From both the ligation patterns in (B) and Supplementary Figure 5, we know that the upper product is favored over the lower product. An asterisk indicates the position of the radioisotope label. (B) In each reaction, 20 nM substrate was incubated with the protein(s) indicated above in a 10 μl reaction for 30 min at 37°C. After incubation, reactions were deproteinized and analyzed by 8% denaturing PAGE. Protein concentrations: Ku, 25 nM; X4-LIV, 50 nM; pol mu or lambda, 25 nM. Twenty-five micromolars of each dNTP were added to the reaction as indicated. ATP (100 μM) was also added in indicated reactions. ‘M’ indicates 50 bp DNA ladder. Download figure Download PowerPoint Surprisingly, even an –AGC 3′ overhang on the top strand can ligate to a GA– 3′ overhang end to form a small, but obvious amount of dimer (Figure 4B, left panel, lane 4). This is unanticipated because the C of the –AGC must ligate across a 1 nt gap to achieve ligation. The amount of ligation was lower than that seen for the –AGT substrate above, perhaps because of the difficulty in ligating across the 1 nt gap (Figure 4B, compare left panel, lane 4 versus right panel, lane 4). The efficiency of the ligation for the –AGT substrate explains the large number of products in which a T is added when the overhang is –AG (Figure 3C, upper, junction sequence a versus b–e). These studies confirm that random addition of a single complementary nucleotide is sufficient to support ligation, and suggests that this occurs even if this ligation must occur across a 1 nt gap. When we tested a corresponding amount of pol lambda relative to pol mu, as assessed by template-dependent synthesis (Supplementary Figure 4), pol lambda supported only a low level of ligation (Figure 3B, lane 2). We sequenced the small number of junctions formed (from Figure 3B, lane 2, dimer position), and these had T, CT, or GT additions, consistent with a very low level of template-independent addition, some of which provided 1 bp of terminal microhomology. XRCC4:DNA ligase IV can ligate across gaps To test formally whether XRCC4:DNA ligase IV could ligate across a gap, we examined substrate ligation by this ligase complex with or without other components (Figure 5). The XRCC4:DNA ligase IV complex alone could ligate a 4 bp terminal microhomology with a 1 nt gap in the ligatable strand (Figure 5B, left panel, lane 2). This ligase complex could also ligate across a 1 nt gap using a 2 bp terminal microhomology (Figure 5B, right panel, lane 2 and Supplementary Figure 6, lane 4). Sequencing of junctions confirmed the ligation across gaps (Figure 5C). Figure 5.XRCC4:DNA ligase IV can ligate over a gap. (A) Two substrates with different lengths of terminal microhomology for ligation were designed to test the direct ligation over a gap by XRCC4:DNA ligase IV. There is a one-nucleotide gap on the ligatable strand. Only the favored joining product is shown under each substrate. An asterisk indicates the position of the radioisotope label. (B) Reactions were performed as in Figure 4B, except that all the reactions include 100 μM of ATP. (C) Dimer products from the selected lanes were cut out of the gel, extracted, and then PCR amplified, TA-cloned, and sequenced. The junction sequences for the ligatable strand were provided. Download figure Download PowerPoint Hence, the XRCC4:DNA ligase IV can support ligation across gaps in the absence of other proteins. Ku stimulated the ligation across a gap (Figure 5B, left panel, lane 3, and right panel, lanes 4 and 5). Addition of pol lambda or pol mu further stimulated the ligation (Figure 5B, left panel, lanes 4–7), but this was because addition of T occurred (sometimes along with additional nucleotides), as documented by sequencing (Figure 5C, left, sequences from lanes 4 to 7, or right, sequences from lanes 7 and 8; Supplementary Figure 6). The rate of ligation of ends that have gaps adjacent to 2 bp of terminal microhomology is roughly 10 times as slow as the rate of ligation of a nick adjacent to a 4 bp block of terminal microhomology (Supplementary Figure 7). XRCC4:DNA ligase IV can ligate fully incompatible DNA ends We were interested in the minimal amount of terminal microhomology needed to support ligation (Figure 6A). We found that, in some cases, Ku and XRCC4:DNA ligase IV were sufficient to complete the ligation of entirely incompatible DNA ends (Figure 6B, lanes 4 and 5 versus 1–3). Sequences of the dimer species confirmed that the ligation occurred with no alteration of either DNA end (see Figure 6C, sequences from the dimer ligation product in lanes 4 and 5). Figure 6.XRCC4:DNA ligase IV and Ku can ligate fully incompatible DNA ends. (A) A substrate without any homology for ligation was designed on the basis of the second substrate in Figure 5A to test the ligation with XRCC4:DNA ligase IV. Two alternative joining pathways are proposed next to the substrate. An asterisk indicates the position of the radioisotope label. (B) Reactions were performed as in Figure 4B, except that all the reactions include 100 μM of ATP. In lane 7, the band below the dimer product is most likely the hairpin structure of the dimer product that is ligated in the manner we proposed in (A), second product. (C) Dimer products from the selected lanes were cut out of the gel, extracted, and then PCR amplified, TA-cloned, and sequenced. The junction sequences for the ligatable strand were provided. Download figure Download PowerPoint In addition to the ligation of this incompatible DNA end pair, we also observed that a DNA end with a 3′ overhanging A could be ligated to an end with a 3′ overhanging –AGC (data not shown). Another example of the ligation of two incompatible ends is the joining of two DNA ends, each with a 3′ A overhang (Supplementary Figure 8). Hence, Ku and XRCC4:DNA ligase IV can ligate a subset of fully incompatible ends that do not share even 1 bp of terminal microhomology. The rate of ligation of DNA ends that are incompatible with no terminal microhomology is obviously much slower than for ends that are stabilized by tw" @default.
• W2053016947 created "2016-06-24" @default.
• W2053016947 creator A5029291507 @default.
• W2053016947 creator A5055153364 @default.
• W2053016947 creator A5064698650 @default.
• W2053016947 creator A5079559642 @default.
• W2053016947 creator A5081248010 @default.
• W2053016947 creator A5085922344 @default.
• W2053016947 date "2007-02-08" @default.
• W2053016947 modified "2023-10-01" @default.
• W2053016947 title "XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps" @default.
• W2053016947 cites W1532833901 @default.
• W2053016947 cites W1627029737 @default.
• W2053016947 cites W1966254852 @default.
• W2053016947 cites W1967653717 @default.
• W2053016947 cites W1972821008 @default.
• W2053016947 cites W1974446138 @default.
• W2053016947 cites W1975096626 @default.
• W2053016947 cites W1977145510 @default.
• W2053016947 cites W1979568780 @default.
• W2053016947 cites W1982305908 @default.
• W2053016947 cites W1997056099 @default.
• W2053016947 cites W2001784394 @default.
• W2053016947 cites W2008181290 @default.
• W2053016947 cites W2011320725 @default.
• W2053016947 cites W2011522975 @default.
• W2053016947 cites W2012425247 @default.
• W2053016947 cites W2024144876 @default.
• W2053016947 cites W2034746516 @default.
• W2053016947 cites W2034925688 @default.
• W2053016947 cites W2037342945 @default.
• W2053016947 cites W2037517612 @default.
• W2053016947 cites W2046561854 @default.
• W2053016947 cites W2051148032 @default.
• W2053016947 cites W2052424743 @default.
• W2053016947 cites W2067982379 @default.
• W2053016947 cites W2073403193 @default.
• W2053016947 cites W2088411674 @default.
• W2053016947 cites W2088698677 @default.
• W2053016947 cites W2089760420 @default.
• W2053016947 cites W2092503176 @default.
• W2053016947 cites W2096630319 @default.
• W2053016947 cites W2099458703 @default.
• W2053016947 cites W2100518470 @default.
• W2053016947 cites W2110210919 @default.
• W2053016947 cites W2114939957 @default.
• W2053016947 cites W2116718924 @default.
• W2053016947 cites W2126077438 @default.
• W2053016947 cites W2135735491 @default.
• W2053016947 cites W2148760839 @default.
• W2053016947 cites W2150661602 @default.
• W2053016947 cites W2154849596 @default.
• W2053016947 cites W2155375991 @default.
• W2053016947 cites W2161187503 @default.
• W2053016947 cites W2163658254 @default.
• W2053016947 cites W2170482532 @default.
• W2053016947 cites W2477626371 @default.
• W2053016947 doi "https://doi.org/10.1038/sj.emboj.7601559" @default.
• W2053016947 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1933392" @default.
• W2053016947 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17290226" @default.
• W2053016947 hasPublicationYear "2007" @default.
• W2053016947 type Work @default.
• W2053016947 sameAs 2053016947 @default.
• W2053016947 citedByCount "155" @default.
• W2053016947 countsByYear W20530169472012 @default.
• W2053016947 countsByYear W20530169472013 @default.
• W2053016947 countsByYear W20530169472014 @default.
• W2053016947 countsByYear W20530169472015 @default.
• W2053016947 countsByYear W20530169472016 @default.
• W2053016947 countsByYear W20530169472017 @default.
• W2053016947 countsByYear W20530169472018 @default.
• W2053016947 countsByYear W20530169472019 @default.
• W2053016947 countsByYear W20530169472020 @default.
• W2053016947 countsByYear W20530169472021 @default.
• W2053016947 countsByYear W20530169472022 @default.
• W2053016947 countsByYear W20530169472023 @default.
• W2053016947 crossrefType "journal-article" @default.
• W2053016947 hasAuthorship W2053016947A5029291507 @default.
• W2053016947 hasAuthorship W2053016947A5055153364 @default.
• W2053016947 hasAuthorship W2053016947A5064698650 @default.
• W2053016947 hasAuthorship W2053016947A5079559642 @default.
• W2053016947 hasAuthorship W2053016947A5081248010 @default.
• W2053016947 hasAuthorship W2053016947A5085922344 @default.
• W2053016947 hasBestOaLocation W20530169471 @default.
• W2053016947 hasConcept C175114707 @default.
• W2053016947 hasConcept C54355233 @default.
• W2053016947 hasConcept C552990157 @default.
• W2053016947 hasConcept C86803240 @default.
• W2053016947 hasConceptScore W2053016947C175114707 @default.
• W2053016947 hasConceptScore W2053016947C54355233 @default.
• W2053016947 hasConceptScore W2053016947C552990157 @default.
• W2053016947 hasConceptScore W2053016947C86803240 @default.
• W2053016947 hasIssue "4" @default.
• W2053016947 hasLocation W20530169471 @default.
• W2053016947 hasLocation W20530169472 @default.
• W2053016947 hasLocation W20530169473 @default.
• W2053016947 hasLocation W20530169474 @default.
• W2053016947 hasOpenAccess W2053016947 @default.

• next