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Comparative Study
. 2007 Feb 21;26(4):1010-23.
doi: 10.1038/sj.emboj.7601559. Epub 2007 Feb 8.

XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

Jiafeng Gu  1 , Haihui Lu, Brigette Tippin, Noriko Shimazaki, Myron F Goodman, Michael R Lieber
Affiliations
Comparative Study

XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

Jiafeng Gu et al. EMBO J. .

Erratum in

  • EMBO J. 2007 Jul 25;26(14):3506-7

Abstract

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.

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Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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.
Figure 5
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.
Figure 6
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.
Figure 7
Figure 7
Ligase activity comparison with DNA double-strand break substrates. (A) JG*161/162 is a substrate with a nick on the ligatable strand. JG*162/161 is a substrate with a 1 nt gap on the ligatable strand and has 4 bp of terminal microhomology for ligation. JG*163/166 is a substrate with a 1 nt gap on the ligatable strand and has a 2 bp terminal microhomology for ligation. JG*163/186 is a substrate with fully incompatible ends for ligation. A star indicates the position of the radioisotope label. (B), (C), and (D), 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; human ligase I, human ligase III, and T4 DNA ligase were first normalized to the same activity of 50 nM X4-LIV using a single-strand nick substrate, and then used in equivalent amounts of activity for the reactions shown. One millimolar of ATP was also added in specified reactions. ‘M' indicates a 50 bp DNA ladder. Ligase abbreviations: I, human ligase I; III, human ligase III; IV, human XRCC4-ligase IV; T4, T4 DNA ligase.
Figure 8
Figure 8
Function of XRCC4:DNA ligase IV in ligating incompatible DNA ends. (A) Ku and XRCC4:DNA ligase IV can ligate fully incompatible DNA ends. XRCC4:DNA ligase IV randomly bind to one DNA end, but this interaction is less stable than if Ku is also bound. Ku may stimulate the interaction by increasing the occupancy time of XRCC4:DNA ligase IV at the DNA end. When another DNA end comes close to this complex, XRCC4:DNA ligase IV binds it and ligates the two ends. (B) XRCC4:DNA ligase IV alone can ligate across a gap. XRCC4:DNA ligase IV randomly binds to one DNA end (although this interaction is not as stable as when Ku is present). The 2 bp of terminal microhomology between the DNA ends increases the chance for XRCC4:DNA ligase IV to ligate. (C) Template-independent polymerase activity of pol mu creates terminal microhomology for ligation by XRCC4:DNA ligase IV. Pol mu can add nucleotides to the DNA end in its template-independent mode. A 1 bp terminal microhomology between the DNA ends (‘t' in this example) permits annealing of the ends and improves the efficiency of ligation.
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