Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence

Abstract

The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg2+, but only of incompatible DNA ends at higher concentrations of Mg2+, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles.

Figure 1.

DNA substrates with a gap are ligated by XRCC4-Ligase IV in a sequence-dependent manner. (A) Three 73 bp substrates with different 3′ overhangs were designed and tested for the efficiency of ligation over a gap by XRCC4-Ligase IV. A star indicates the position of the radioisotope label. JG*163/166 and JG*166/163 are the same duplex DNA substrate but with one strand labeled or the other. In the ligations, a dimer, e.g. involves two molecules of the same species joined head to tail. (B) In each reaction, 20 nM substrate was incubated with the protein(s) indicated above the lane in a 10 μl reaction for 30 min at 37°C. After incubation, reactions were deproteinized and analyzed by 8% denaturing PAGE. Protein concentrations are as follows: Ku, 25 nM; XRCC4-Ligase IV, 50 nM; pol mu, 25 nM; T4 DNA ligase, 120 nM; XLF, 100 nM. Twenty-five micromolar of each dNTP was added to the reactions where pol mu was present. Hundred micromolar of ATP was also added in each reaction shown in the left panel, and 1 mM of ATP was added to the reactions where T4 DNA ligase was present. ‘M’ indicates 50 bp DNA ladder. Ligation products were quantified, and ligation efficiencies were provided under each lane. DNA sequence analysis confirmed that the junctional sequences conformed to those shown in A. Note that when the top strand is assayed for ligation, the bottom strand has an unligatable 5′OH at the junction, and likewise when the bottom strand is assayed for ligation, the top strand has an unligatable 5′OH at the junction. If we do permit ligation of both strands, then the more readily ligated strand now markedly facilitates the ligation of the anti-parallel strand (unpublished data). The same results for the right panel are obtained when a lower amount of ligase is used (data not shown).

Figure 2.

XLF stimulates incompatible end ligation by XRCC4-Ligase IV but not compatible and blunt end ligations. (A) Substrates with compatible, incompatible 3′ overhangs and blunt ends were designed and tested for their ligation efficiency with or without XLF. A star indicates the position of the radioisotope label. In the ligations, a dimer, e.g. involves two molecules of the same species joined head to tail. (B and C), In each reaction, 20 nM substrate was incubated with the protein(s) indicated above the lane 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; XLF, 100 nM. ‘M’ indicates 50 bp DNA ladder. DNA sequence analysis confirmed that the junctional sequences conformed to those shown in A.

Figure 3.

XLF stimulation of ligation as a function of Mg²⁺ concentration. (A and B) Compatible substrate JG*163/166 and incompatible substrate JG*163/186 were tested for the XLF stimulation at various Mg²⁺ concentrations. In each reaction, 20 nM substrate was incubated with 25 nM Ku, 50 nM XRCC4-Ligase IV and with or without 100 nM XLF in a 10 μl reaction with Mg²⁺ concentrations varying from 0 to 20 mM. For substrate JG*163/166, reactions were carried out at 37°C for 2.5 min; for substrate JG*163/186, reactions were carried out at 37°C for 30 min. After incubation, reactions were deproteinized and analyzed by 8% denaturing PAGE. (C), Ligation products were quantified and ligation efficiencies were provided along with each substrate under different Mg²⁺ concentrations. The fold of XLF stimulation was calculated.

Figure 4.

Single-stranded DNA can be ligated by XRCC4-Ligase IV. Single-stranded 30 dT substrate (JG*169) was tested for direct ligation by XRCC4-Ligase IV. In each reaction, 50 nM substrate was incubated with different amounts of XRCC4-Ligase IV as indicated above each lane in a 10 μl reaction for 30 min at 37°C. After incubation, reactions were deproteinized and analyzed by 10% denaturing PAGE. ‘M’ indicates 50 bp DNA ladder. Quantification shows that 0.1% of the substrate is converted to the ligated products in lanes 6–8. No ATP is present in the incubations, consistent with the pre-charged status of DNA ligase IV. [The amount of material in the well is small relative to the amount of substrate and does not vary linearly with the amount of ligase (compare lanes 3 versus 7).]