DNA end-joining catalyzed by human cell-free extracts

GM00558, GM09820, and GM06315 were obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). They were grown in suspension to a density of 8 × 10⁵ cells/ml and were harvested and washed once in DMEM containing 10% fetal calf serum, three times in ice-cold PBS, and once in hypotonic lysis buffer (10 mM Tris⋅HCl, pH 8.0/1 mM EDTA/5 mM DTT). Cells were resuspended in 2 vol of hypotonic buffer and, after 20 min at 0°C, were lysed by homogenization (20 strokes with an “A” pestle), and protease inhibitors were added (phenylmethylsulfonyl fluoride, 0.17 mg/ml; aprotinin, 0.01 trypsin inhibitor units/ml; pepstatin, 1 μg/ml; chymostatin, 1 μg/ml; leupeptin, 1 μg/ml). After 20 min on ice, 0.5 vol of high salt buffer (50 mM Tris⋅HCl, pH 7.5/1 M KCl/2 mM EDTA/2 mM DTT) was added, and the extract was centrifuged for 3 hours at 42,000 rpm in a Beckman SW50.1 rotor. The supernatant was dialyzed for 3 hours against E buffer [20 mM Tris⋅HCl, pH 8.0/0.1 M KOAc/20% (vol/vol) glycerol/0.5 mM EDTA/1 mM DTT] and was fast-frozen and stored at −70°C.

For fractionation studies, GM00558 extract (10 mg) was loaded onto a 1-ml phosphocellulose column equilibrated in E buffer, and proteins were eluted by using E buffer containing 0.15, 0.5, or 0.9 M KCl. Peak fractions (0.5 ml) designated PC-A (column flow through; 3.4 mg/ml), PC-B (0.15 M step; 1.8 mg/ml), PC-C (0.5 M step; 2.2 mg/ml), and PC-D (0.9 M step; 0.34 mg/ml) were dialyzed against E buffer.

Xrcc4/ligase IV (MonoQ fraction 11; ref. 22), DNA ligase III (33), and DNA-PK (8) generously were provided by the laboratories of T. Lindahl (Imperial Cancer Research Fund) and S. P. Jackson (Cambridge University). Form I pDEA-7Z duplex plasmid DNA (3.0 kilobases) (34), was linearized with BsaI (NEB), was dephosphorylated, and was 5′-³²P-labeled by using polynucleotide kinase. Uniformly ³²P-labeled form I pFB585 DNA (7.7 kilobases) was prepared by growth in the presence of [³²P]orthophosphate.

Reactions (10 μl) were carried out in 50 mM triethanolamine⋅HCl (pH 7.5), 0.5 mM Mg(OAc)₂, 60 mM KOAc, 2 mM ATP, 1 mM DTT, and 100 μg/ml BSA. Cell-free extract was incubated for 5 min at 37°C before the addition of ³²P-labeled DNA (10 ng). Incubation was for 1 hour at 37°C. ³²P-labeled DNA products were deproteinized and analyzed by electrophoresis through 0.6% agarose gels followed by autoradiography. Quantification of joining efficiency was carried out by phosphorimaging. Experiments with the DNA-PK inhibitors wortmannin or LY294002 (Sigma) were carried out by incubation of the inhibitor with extract for 30 min on ice before a shift to 37°C (10 min) and addition of ³²P-labeled DNA. Incubation then was continued for 1 hour.

Rabbit polyclonal sera against Xrcc4 (3), ATM (35), and DNA ligase IV (S. Critchlow and S. P. Jackson, personal communication) were provided by S. P. Jackson. Antisera against DNA ligases I (36) and III (37) were provided by T. Lindahl, and those against Ku70, Ku86, and DNA-PK_cs were purchased from Serotec. Purified antibodies against DNA-PK_cs used for Western blotting were from Calbiochem.

For immunodepletion, GM00558 extract (50 μg) was incubated on a rotary wheel with polyclonal antiserum (3 μl) at 4°C for 90 min (50 μl total volume). The extract/antibody mixture was added to protein A-Sepharose beads and was incubated with rotation for 60 min. The beads then were removed by repeated centrifugation. Western blotting was carried out by standard procedures, and proteins were visualized by ECL (Amersham).

Nonhomologous end-joining catalyzed by extracts of human lymphoblastoid cell line GM00558 was observed by the ligation of 5′-³²P-end-labeled linear duplex DNA (Fig. 1A, lanes 3 and 4). Approximately 10–20% of the input DNA molecules were ligated during a 1-hour incubation in a reaction that depended on the presence of ATP and Mg²⁺. End-joining activity was observed with extracts prepared from a range of lymphoblastoid cell lines (Fig. 1B), although some extracts (e.g., GM06315; see Fig. 1B, lanes 6 and 7) showed higher levels of nuclease activity, and end-joining could not be analyzed.

Double-strand breaks containing 3′-overhangs provided the best substrate for rejoining, as seen by comparing the ability of extract to rejoin linear DNA containing 5′-overhangs, blunt ends, or 3′-overhangs (Fig. 1C). Indeed, 27% of the 3′-overhang substrate was converted into dimers (Fig. 1C, lane 4), compared with 13% of the 5′-overhangs (Fig. 1C, lane 2) and 6% of the blunt-ended substrate (Fig. 1C, lane 3). The ligated products could be recut with the same restriction enzyme (Fig. 1D, lanes 2, 4 and 6), indicating that repair occurred without loss or addition of nucleotide sequences at the joint. Only in the case of the 3′-substrate was a fraction (≈5%) of the product resistant to cleavage (Fig. 1D, lane 6).

To determine whether the DNA end-joining activity could be attributed to a specific DNA ligase, extracts prepared from GM00558 were preincubated with polyclonal antisera raised against human DNA ligase I, ligase III, or ligase IV before addition of the ³²P-end-labeled linear DNA and subsequent incubation. We found that antisera against DNA ligase IV inhibited end-joining (Fig. 2A, lanes 11–14) whereas antisera against either ligase I (Fig. 2A, lanes 3–6) or ligase III (Fig. 2A, lanes 7–10) did not.

Because DNA ligase IV associates tightly with Xrcc4 (3, 4), we analyzed whether antiserum against Xrcc4 also could inhibit ligation. Fig. 2B shows that the anti-Xrcc4 serum abolished end-joining activity (Fig. 2B, lanes 7–10) whereas preimmune serum did not (Fig. 2B, lanes 3–6). None of the antisera used above affected the activity of T4 DNA ligase.

To confirm the requirement for Xrcc4, antiserum was used to immunodeplete Xrcc4 from the extract. As predicted, the Xrcc4-immunodepleted extract failed to promote end-joining (Fig. 2D, lane 3). Because Xrcc4/ligase IV complexes are highly stable (3, 4), immunodepletion also removed the majority of the ligase IV (Fig. 2C, compare lanes 1 and 2). Activity was reconstituted by the addition of purified Xrcc4/ligase IV complex (Fig. 2D, lane 4). Thus, DNA ligase IV and Xrcc4 are required for DNA end-joining catalyzed by human cell-free extracts. However, ligase IV/Xrcc4 were insufficient for the reaction because they failed to promote end-joining in the absence of extract (Fig. 2D, lane 5).

Antisera against the three subunits of DNA-PK (Ku70, Ku86, and DNA-PK_cs) also reduced end-joining activity (Fig. 3A, lanes 4–12) whereas a control serum (anti-ATM) had no effect (Fig. 3A, lanes 1–3). Inhibition was, however, less than complete, because of either the large size of DNA-PK (>600 kDa) or the abundance of DNA-PK within the cell (38). Inhibitors of DNA-PK_cs such as wortmannin (9) and LY294002 (G. M. Smith and S. P. Jackson, personal communication) also blocked DNA end-joining in vitro (Fig. 3 B and C).

To determine whether factors other than those described above were required for DNA end-joining in vitro, the extract was fractionated by phosphocellulose chromatography. The unbound fraction (PC-A) and proteins that bound to the resin and eluted with three steps of increasing salt concentration (designated PC-B, PC-C, and PC-D) were tested for end-joining activity. Fraction PC-C promoted low levels of end-joining (Fig. 4A, lane 5) whereas the others did not (Fig. 4A, lanes 3, 4, and 6). When fractions PC-A and PC-C were mixed, however, efficient end-joining activity was reconstituted such that the specific activity was greater than that of the unfractionated extract (Fig. 4B, lanes 2–4). As expected, the reconstituted activity depended on DNA-PK and Xrcc4/ligase IV, as shown by using LY294002 and anti-Xrcc4 antiserum.

Because PC-A and PC-C were sufficient for end-joining in vitro, each fraction was analyzed by immunoblotting for the presence of DNA-PK, ligase IV, and Xrcc4. Fraction PC-C contained Ku70, Ku86, DNA-PK_cs, DNA ligase IV, and Xrcc4, although the majority of the Ku70/86 heterodimer was found in PC-B (Fig. 4D). DNA-dependent kinase activity was detected in fraction PC-C (Fig. 4C). Because substantial amounts of Ku70, Ku86, DNA-PK_cs, DNA ligase IV, and Xrcc4 were in PC-C, the requirement for PC-A for end-joining was surprising and suggestive of a need for as yet unidentified factor(s). Whether these factor(s) are specific for DNA-PK-dependent end-joining is presently unknown, although specificity is indicated by observations showing that PC-A failed to stimulate end-joining by T4 DNA ligase or human DNA ligase III.

During these fractionation studies, we observed additional end-joining activities that were not inhibited by anti-Xrcc4 antibodies or DNA-PK inhibitors (data not shown). Similarly, when cell-free extracts were prepared by other protocols (39), joining activities were observed that did not show the same factor requirements as those reported here. Moreover, we found that rodent extracts promoted highly efficient end-joining reactions that were inhibited only partially by loss of DNA-PK function, either through mutation, antibody inhibition, or the use of chemical inhibitors (data not shown). These end-joining activities may be similar to those reported previously (29–32), and their relationship to DNA-PK-dependent reactions will require further investigation.