Polynucleotide Kinase and Aprataxin-like Forkhead-associated Protein (PALF) Acts as Both a Single-stranded DNA Endonuclease and a Single-Stranded DNA 3′ Exonuclease and Can Participate in DNA End Joining in a Biochemical System

Background: PALF binds to the NHEJ protein XRCC4.

Results: PALF is a single-stranded DNA endonuclease and 3′ exonuclease. PALF can coordinate with known NHEJ proteins to achieve ligation.

Conclusion: In vitro and in vivo, PALF is able to cooperate with NHEJ proteins to join double-stranded DNA breaks.

Significance: A second nuclease, in addition to Artemis, can function with NHEJ proteins to achieve DNA end joining.

Abstract

Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF, also called aprataxin- and PNK-like factor (APLF)) has been shown to have nuclease activity and to use its forkhead-associated domain to bind to x-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4). Because XRCC4 is a key component of the ligase IV complex that is central to the nonhomologous DNA end joining (NHEJ) pathway, this raises the possibility that PALF might play a role in NHEJ. For this reason, we further studied the nucleolytic properties of PALF, and we searched for any modulation of PALF by NHEJ components. We verified that PALF has 3′ exonuclease activity. However, PALF also possesses single-stranded DNA endonuclease activity. This single-stranded DNA endonuclease activity can act at all single-stranded sites except those within four nucleotides 3′ of a double-stranded DNA junction, suggesting that PALF minimally requires approximately four nucleotides of single-strandedness. Ku, DNA-dependent protein kinase catalytic subunit, and XRCC4-DNA ligase IV do not modulate PALF nuclease activity on single-stranded DNA or overhangs of duplex substrates. PALF does not open DNA hairpins. However, in a reconstituted end joining assay that includes Ku, XRCC4-DNA ligase IV, and PALF, PALF is able to resect 3′ overhanging nucleotides and permit XRCC4-DNA ligase IV to complete the joining process in a manner that is as efficient as Artemis. Reduction of PALF in vivo reduces the joining of incompatible DNA ends. Hence, PALF can function in concert with other NHEJ proteins.

Introduction

PALF² (also called APLF, C2orf13, and Xip1) is a recently described nuclease that was named for the fact that it shares properties with polynucleotide kinase (PNK) and aprataxin, hence the name PNK- and aprataxin-like factor (also APLF for aprataxin- and PNK-like factor) (1–4). The shared property is the binding to XRCC4, a key component of the NHEJ ligase complex. PALF, PNK, and aprataxin also all have a forkhead-associated (FHA) domain. The FHA domain is the site of binding to XRCC4 (5). The kinase CK2 phosphorylates XRCC4 (5), and this phosphorylated form of XRCC4 is necessary for the FHA domain of PALF, PNK, and aprataxin to bind XRCC4 (1, 3). PALF has also been reported to bind to Ku (1, 3) as well as DNA-PKcs (1) and is important for cell survival after low-dose ionizing radiation (4).

XRCC4 is a key component of the XLF-XRCC4-DNA ligase IV complex, the primary ligase for vertebrate NHEJ (6, 7). NHEJ is the major pathway for repair of double-strand breaks and includes the factors Ku, DNA-PKcs, Artemis, polymerase μ, and polymerase λ as well. The nuclease activities of NHEJ are thought to be largely provided by Artemis (8). Artemis has endonucleolytic activity at all double- to single-strand transitions (9). For repair of double-strand breaks, 5′ overhangs, 3′ overhangs, and hairpin structures are key substrates of Artemis. Artemis also appears to have 5′ exonuclease activity, although mutagenesis has not yet definitively demonstrated this activity to be intrinsic to Artemis (10, 11). Artemis and DNA-PKcs form a tight complex, and Artemis endonucleolytic activity at double- to single-strand transitions requires activation of DNA-PKcs (8, 12). DNA-PKcs is a serine/threonine protein kinase that is only activated by duplex DNA ends. When DNA-PKcs is activated by duplex DNA ends, it uses ATP to autophosphorylate itself and Artemis (8, 13). The autophosphorylation causes a conformational change in DNA-PKcs (14, 15), and this presumably causes a conformational change in Artemis to account for the activation of the endonucleolytic properties of Artemis (12). When the C-terminal portion of Artemis is removed, Artemis acquires endonucleolytic properties independent of DNA-PKcs (16, 17).

The nucleolytic properties of PALF have been described as a 3′ exonuclease and a double-stranded DNA endonuclease (1). PALF was also found to have nicking activity against various DNA molecules containing abasic sites (1). Here we confirm the 3′ exonuclease activity of PALF (1). The endonucleolytic properties of PALF appear to be more general than described originally. PALF is able to endonucleolytically act on single-stranded DNA. The action described previously at double-stranded DNA ends may simply reflect breathing of those ends into transient single-stranded tails. In contrast to Artemis, PALF is unaffected in its nucleolytic properties by DNA-PKcs. PALF is also unaffected by Ku or by XRCC4-DNA ligase IV. However, at incompatible ends with overhangs, we show that purified PALF is able to function in concert with purified Ku and XRCC4-DNA ligase IV in end joining reactions at an efficiency that is equivalent to Artemis when used in place of PALF. Finally, we find that in vivo siRNA knockdown of PALF results in a significant drop in the joining of incompatible DNA ends. Hence, PALF appears to be capable of participating in end joining with other NHEJ proteins.

EXPERIMENTAL PROCEDURES

Oligonucleotides and DNA Substrates

Oligonucleotides used in this study were synthesized by Operon Biotechnologies, Inc. (Huntsville, AL) and Integrated DNA Technologies, Inc. (San Diego, CA). We purified the oligonucleotides using 12% or 15% denaturing PAGE and determined the concentration spectrophotometrically.

DNA substrate 5′ end labeling was done with [γ-³²P]ATP (3000 Ci/mmol) (PerkinElmer Life Sciences, Boston, MA) and T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's instructions. Substrates were incubated with [γ-³²P]ATP and T4 PNK for 30 min at 37 °C. T4 PNK was subsequently inactivated by incubating samples at 72 °C for 20 min. Unincorporated radioisotope was removed by using G-25 Sephadex (Amersham Biosciences, Inc.) spin-column chromatography. For the hairpin substrate, YM164-labeled oligonucleotide was diluted in a buffer containing 10 mm Tris-hydrochloride (pH 8.0), 1 mm EDTA (pH 8.0), and 100 mm NaCl, heated at 100 °C for 5 min, allowed to cool to room temperature for 3 h, and then incubated at 4 °C overnight.

The sequences of the oligonucleotides used in this study are as follows: JG68, 5′-GAT CCT TCT GTA GGA CTC ATG-3′; JG169, 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3′; YM164, 5′-TTT TTG ATT ACT ACG GTA GTA GCT ACG TAG CTA CTA CCG TAG TAA T-3′; YM130, 5′-TTT TTT TTT TTT TTT ACT GAG TCC TAC AGA AGG ATC-3′; YM149, 5′-ACT GAG TCC TAC AGA AGG ATC TTT TTT TTT TTT TTT-3′; YM8, 5′-AGG CTG TGT TAA GTA TCT GCG CTC GCC CTC AGA GG-3′; YM9, 5′-CCT CTG AGG GCG AGC GCA GAT ACT TAA CAC AGC CT-3′; JG258, 5′-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CGG GG-3′; SL11, 5′-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GCC CCA AAA AA-3′; and SL15, 5′-ACT GAG TCC TAC AGA AGG ATC TTT TTT TTT TTS SSS. The sequence of siRNA for PALF, 5′-CCA GAU GAC UCC CAC AAA UAG, was synthesized, annealed with a complementary strand, and used with a final concentration of 20 μm. Antibody against PALF was prepared as reported previously (1).

Protein Expression and Purification

N-terminal His-tagged PALF cloned into a pET-16b (Novagen) vector has been described previously (1). Soluble His-PALF was expressed and purified from pLysE BL21(DE3)-competent cells (Invitrogen). Cells were precultured in ampicillin until A₆₀₀ of 0.5. Cells were then induced with isopropyl 1-thio-β-d-galactopyranoside (1 mm) and cultured for an additional 3 h before harvesting. Harvested BL21(DE3) cells expressing His-PALF were resuspended in Ni-NTA binding buffer (50 mm NaH₂PO₄ (pH 7.8), 0.5 m KCl, 2 mm β-mercaptoethanol, 10% glycerol, 0.1% Triton X-100, and 20 mm imidazole (pH 7.8)) supplemented with protease inhibitors and lysed by sonication. The cell lysate was applied to a Ni-NTA-agarose resin (Qiagen). Resin was washed with 35 mm imidazole. His-PALF was eluted off with binding buffer plus 500 mm imidazole. Eluted fractions were dialyzed against Hi-Trap heparin binding buffer (50 mm Tris-HCl (pH 7.5), 10% glycerol, 2 mm EDTA, 1 mm DTT, 100 mm NaCl, 0.02% Nonidet P-40), loaded onto a pre-equilibrated Hi-Trap heparin column, and eluted with a linear gradient to 1 m NaCl over 20 ml. We were able to obtain a yield of 200 μg from a 1-liter starting culture. His-PALF-containing fractions were reconcentrated onto Ni-NTA-agarose and eluted in binding buffer plus 500 mm of imidazole to a final elution volume of 200 μl. His-PALF was then applied to a Superose 12 gel filtration column (GE Healthcare) and eluted with 250 mm NaCl, 10% glycerol, 50 mm Tris-HCl (pH 7.5), and 1 mm DTT. PALF samples were stored at 4 °C and used within 2 weeks because the nuclease activity of PALF is highly sensitive to freezing, as pointed out previously (1). The expression and purification of DNA-PKcs from HeLa cells has been described previously (8).

In Vitro Nuclease and Ligation Assays

We extended the PALF nuclease optimizations performed previously (1) by further testing activity in a range of pH (6.5 to 8), divalent cation concentration (0 to 20 mm MgCl₂), and monovalent salt concentration (1 mm to 100 mm KCl). On the basis of this, in vitro DNA nuclease assays were performed in a total volume of 10 μl with a buffer composition of 25 mm Tris-HCl (pH 7.5), 10 mm KCl, 10 mm MgCl₂, 1 mm DTT and 50 ng/μl BSA. In the reaction, 50 nm single-stranded DNA substrate with an overhang (3′ or 5′) or 20 nm hairpin substrate were incubated with 125 nm PALF, and, in specified cases, one or more of the following: 126 nm DNA-PKcs, 100 nm Ku, 75 nm XRCC4/ligase IV, or a combination of the three proteins. In reactions containing XRCC4/ligase IV, the XRCC4 and ligase IV were prephosphorylated by CK2 according to the manufacturer's instructions (Sigma-Aldrich). Unless specified otherwise, when DNA-PKcs was present, 0.5 mm ATP and 0.5 μm 35-bp blunt-end DNA (YM 8/9) were also included in the specified reactions. Reactions were then incubated at 37 °C for 1 h. After incubation, reactions were stopped and analyzed on 12% denaturing PAGE gels.

In vitro ligation assays were performed in a total volume of 10 μl with a buffer composition of 25 mm Tris (pH 7.5), 2 mm DTT, 0.025% Triton X-100, 0.1 mm EDTA, 10% PEG, 50 ng/μl BSA, and 5% glycerol. In the in vitro ligation assay, a two-step reaction was performed. In the reaction, 50 nm double- stranded substrate (SL11/JG25) was incubated with 125 nm PALF, and, in specified cases, one or more of the following: 126 nm DNA-PKcs, 100 nm Ku, 75 nm XRCC4/ligase IV, or a combination of the three proteins. Unless specified otherwise, when DNA-PKcs was present, 0.5 mm ATP and 0.5 μm 35-bp blunt-end DNA (YM 8/9) were also included in the specified reactions. A two-step ligation reaction was carried out in the following order: PALF, substrate, and DNA-PKcs were first added to the reaction and incubated at 37 °C for 30 min. Ligase IV-XRCC4 and Ku were then added, and the reaction was incubated for another 30 min at 37 °C. The reactions were stopped, and DNA was phenol-extracted and analyzed on 8% denaturing PAGE gel. Gels were dried, exposed in a phosphorimager cassette, and scanned. Bands were cut out at the dimer position on ligation gels, and we used TOPO TA to clone this DNA into the Invitrogen pCR2.1-TOPO vector. The insert was subsequently sequenced using a Li-Cor 4200 sequencer, and the junctions were analyzed.

NHEJ Assay in Living Human Cells

As reported previously (20), the I-SceI expression plasmid, pCMV-3xNLS-I-SceI, was introduced by transfection with Lipofectamine 2000 reagent into 1.5 × 10⁵ H1299dA3–1#1 cells harboring two I-SceI sites located 1.3 kb apart. The cells were pretransfected with siRNA for 48 h using Lipofectamine RNAiMAX. For FACS analysis, cells were harvested by trypsinization, washed with PBS, and applied to the FACS Calibur apparatus (BD Biosciences). EGFP-positive cells were counted using the Cellquest software.

RESULTS

PALF has 3′ Single-stranded Exonuclease Activity

Previous data has demonstrated that PALF has endonuclease activity which is dependent on the presence of abasic sites (1). We purified PALF using Ni-NTA, HiTrap heparin, and Superose 12 columns (Fig. 1A) and found it to have nuclease activity across an elution peak in proportion to the amount of protein visualized using Coomassie Blue staining (Fig. 1, A and B).

FIGURE 1.

Purification of PALF. A, PAGE gel on Superose 12 fractions. After Ni-NTA and Hi-Trap heparin purification, Superose 12 fractions of PALF are shown on an 8% SDS-PAGE gel stained with Coomassie Blue on which PALF has a gel mobility position at 81 kDa. Ladder designates the protein marker lane, and the fraction numbers are above each lane. B, nuclease activity of PALF corresponding to Superose 12 fractions. Fractions across the Superose 12 elution peak were assayed for nuclease activity using poly(dT) substrate (JG169). Each reaction consists of 50 nm single-stranded DNA substrate (JG169) and 50 nm PALF. Reactions were incubated for 2 h at 37 °C. After incubation, reactions were stopped and analyzed by 12% denaturing PAGE.

Analysis of individual domains of PALF shows that the C-terminal region, spanning amino acids 360–511, is necessary and sufficient to impart single-strand nuclease activity on the protein (supplemental Figs. 1 and 2). The amino acid 360–511 fragment alone contains all of the nucleolytic activity of the PALF protein (supplemental Fig. 1). A truncation mutant lacking the amino acid 378–511 domain has no detectable nuclease activity in our assay (supplemental Fig. 2). The amino acid 378–511 region contains two CYR (cysteine-tyrosine-arginine) motifs, also called PBZ motifs (poly [ADP-ribose] binding zinc finger).

We tested the activity of PALF on a single-stranded DNA substrate to determine whether PALF has nuclease capabilities beyond those described originally. We designed a single- stranded poly dT 30mer labeled at the 5′ end to investigate this (Fig. 2A, lane 1). On this substrate, PALF generates a series of cleavage products (Fig. 2A, lane 2). The addition of DNA-PKcs and ATP does not increase or decrease the nuclease activity of PALF on single-stranded DNA (Fig. 2A, lane 3). Stimulation of DNA-PKcs autophosphorylation by the addition of unlabeled double-stranded DNA, YM8/YM9, also did not affect the activity of PALF (Fig. 2A, lane 4).

FIGURE 2.

3′ to 5′ exonuclease activity of PALF on single-stranded DNA. A, exonuclease activity of PALF on single-stranded DNA. In the reaction, 50 nm single-stranded DNA substrate (JG169) was incubated with the protein(s) indicated above the lane in a 10-μl reaction for 60 min at 37 °C. After incubation, reactions were stopped and analyzed by 12% denaturing PAGE. Protein concentrations are as follows: PALF, 125 nm and DNA-PKcs, 126 nm. As specified, 0.5 mm ATP and 0.5 μm YM8/9 were also included in designated reactions. YM8/9 is a 35-bp blunt-ended double-stranded DNA that is used as DNA-PKcs cofactor. B, 3′ exonuclease monitored with 3′-labeled substrate. In the reaction, 50 nm single-stranded DNA substrate (JG169) labeled at its 3′ end was incubated with PALF in a 10-μl reaction for 60 min at 37 °C. Concentrations are as follows: 250 nm PALF (lane 2) and 125 nm PALF (lane 3). After incubation, reactions were stopped and analyzed by 12% denaturing PAGE. The asterisk represents the radiolabel in all figures. The bold arrow on the DNA substrate diagram beside the gel represents the site of DNA cleavage in all figures.

Artemis was used to generate a ladder starting at the 1 nucleotide position (Fig. 2A, lane 5). PALF does not have any 5′ exonuclease activity. Unlike Artemis, no distinct product is formed at the 1 nucleotide position (Fig. 2A, lane 2 versus lane 5). The nucleolytic cleavage products that we do see can be explained by a combination of 3′ exonuclease activity and single-stranded endonuclease activity. In the presence of Mn²⁺, Artemis does not require DNA-PKcs to function endonucleolytically (10, 17). MnCl₂ does not affect the activity of PALF (Fig. 2A, lane 6).

To differentiate between these two activities, we labeled the same substrate at the 3′ end (Fig. 2B, lanes 1–3). Products generated by PALF from this substrate include a prominent band at the 1 nucleotide position (Fig. 2B, lanes 2 and 3), demonstrating definitive 3′ exonuclease activity. However, a full range of larger products with a weaker profile is also present (Fig. 2B, lanes 2 and 3). The amount of each species in this distribution of products increases relatively equally as the amount of enzyme was increased (twice as much PALF in lane 2 than in lane 3). Given that the substrate was labeled at the 3′ terminus, these weaker products can only be generated by the endonuclease activity of PALF acting on the single-stranded substrate. Hence, we conclude that PALF has endonuclease activity on single-stranded DNA in addition to 3′ exonuclease activity.

PALF Has Single-stranded DNA Endonuclease Activity

If PALF has both 3′ exonuclease activity and single-stranded endonuclease activity, then the endonuclease activity could potentially be masked on a single-stranded DNA substrate. To test for endonuclease activity that does not require substrates with abasic sites, we designed substrates containing either a 15-nucleotide 3′ overhang or a 14-nucleotide 5′ overhang, both labeled at the 5′ end (Fig. 3A, lane 1 for the 5′ overhang substrate, and B, lane 1 for the 3′ overhang substrate). This endonuclease activity cleaves randomly within the 5′ overhang. Unlike Artemis, the addition of DNA-PKcs does not further stimulate the endonuclease activity (Fig. 3a, lane 2 versus lane 3). Addition of unlabeled dsDNA to DNA-PKcs (to stimulate autophosphorylation) also has no effect on the activity of PALF at the 5′ overhang (Fig. 3a, lane 2 versus lane 3). Hence, we conclude that unlike Artemis, PALF does not require DNA-PKcs for endonuclease activity at a 5′ overhang.

FIGURE 3.

Endonuclease activity of PALF on overhangs. A, in specified reactions, 50 nm 5′-labeled double-stranded DNA substrate, YM130/YM68 (5′ overhang), was incubated with 125 nm PALF, 126 nm DNA-PKcs, 0.5 mm ATP, and 0.5 μm YM8/9 in a 10-μl reaction for 60 min at 37 °C. After incubation, reactions were stopped and analyzed on 12% denaturing PAGE. B, in specified reactions, 50 nm 5′-labeled double-stranded DNA substrate, YM149/YM68 (3′ overhang), was incubated with 125 nm PALF, 126 nm DNA-PKcs, 0.5 mm ATP, and 0.5 μm YM8/9 in a 10-μl reaction for 60 min at 37 °C. After incubation, reactions were stopped and analyzed by 12% denaturing PAGE.

Because of its ability to bind to Ku through an intermediate domain between the FHA domain and the CYR motif (poly [ADP-ribose] binding zinc finger motif) of PALF (1, 3), we tested whether the addition of Ku has an effect on nuclease activity of PALF at a 5′ overhang. The addition of Ku along with DNA-PKcs does not appear to modify the activity of PALF activity (data not shown).

Earlier, we saw that the single-stranded DNA endonuclease activity of PALF is less efficient when 5′ labeled single-stranded DNA substrates are cleaved to lengths smaller than 4 nt. The single-stranded endonuclease activity also does not extend into the double-stranded portion of the DNA. This is indicated by the relative lack of bands above the 14th nucleotide, counting from the bottom of the gel (Fig. 3A, lane 2). (The very weak bands present between the 14th and 18th nucleotide may be due to PALF action at sites of breathing of the single strand/double strand DNA junction.) Hence, PALF does not appear to have significant activity on double-stranded DNA.

On a 5′-labeled duplex DNA substrate with a 15-nt poly T 3′ overhang, nine to ten distinct products are seen (Fig. 3B, lane 2). PALF demonstrates endonuclease activity but does not cleave at positions within 4 or 5 nts from the double-stranded portion of the substrate (Fig. 3B, lane 2). The 3′ overhang activity is not stimulated or blocked by the addition of DNA-PKcs and ATP (Fig. 3B, lane 3). Further addition of unlabeled double-stranded DNA, YM8/YM9, with DNA-PKcs also had no effect on the nuclease activity of PALF (Fig. 3B, lane 3). The addition of Ku did not yield any changes when compared with the basal level with PALF alone (data not shown). Like the 5′ overhang substrate, no one cleavage product is dominant over any other. Hence, the nuclease activity on the single-stranded 3′ overhang appears to be random in its position. PALF also has 3′ exonuclease activity. Therefore, the activity we see at the 3′ overhang is a combination of both endonuclease as well as 3′ exonuclease activity on single-stranded regions. When we position four phosphothioester linkages at the 3′ end of the substrate, much less product derives from the 3′ overhang, indicating diminished 3′ exonuclease activity (supplemental Fig. 3, lane 2 versus lane 4). However, some product still arises, attributable to the endonuclease activity, which can nick upstream of the phosphothioester linkages.

For 3′ overhang substrates, PALF seems to functionally occupy approximately four or five nucleotides 3′ to the duplex portion of the substrate, as indicated by termination of nicking at length 26 nt (rather than 21 nt) (Fig. 3B). The approximately four or five nucleotides may reflect the minimum size required for PALF to bind to single-stranded DNA and act upon substrates. This is the same behavior as is seen for Artemis endonuclease action on 3′ overhangs (Fig. 4, lane 5, and Ref. 8). We conclude that PALF has single-stranded endonuclease activity and that this is distinct from its 3′ exonuclease activity.

FIGURE 4.

Lack of endonuclease activity of PALF on hairpin substrates. In specified reactions, 20 nm hairpin DNA substrate, YM164, was incubated with 125 nm PALF, 50 nm Artemis, 126 nm DNA-PKcs, 0.5 mm ATP, and 0.5 μm YM8/9 in a 10-μl reaction for 60 min at 37 °C. After incubation, reactions were stopped and analyzed by 12% denaturing PAGE.

Because of the fact that PALF has endonuclease activity, we further investigated the possibility that PALF could potentially open a hairpin substrate like Artemis. A hairpin substrate with a 20-nucleotide duplex portion and a 6-nucleotide 5′ overhang was used for this experiment (Fig. 4, lane 1). When PALF alone is incubated with the substrate, four endonuclease products can be seen at the bottom of the gel (Fig. 4, lane 2). This is consistent with the endonuclease activity we observed before for the 5′ overhang substrate, YM130/YM68. Once again we used Artemis-DNA-PKcs to generate a ladder to show the 1 nucleotide position and the hairpin opening product at the 28-nt position (Fig. 4, lane 5). This product is absent in the reaction where PALF alone is added (Fig. 4, lane 2). The hairpin opening activity of Artemis is dependent upon DNA-PKcs, and we explored this possibility with respect to PALF. DNA-PKcs and ATP were added to the reaction in addition to PALF. This did not stimulate PALF to cleave the hairpin, and no additional endonuclease activity is seen (Fig. 4, lane 3). Further stimulation of DNA-PKcs by unlabeled DNA YM8/9 also did not stimulate PALF to cleave a hairpin substrate (Fig. 4, lane 4). Therefore, unlike Artemis, PALF does not show DNA-PKcs-dependent ability in opening hairpin substrates (Fig. 4, lane 4 versus lane 5). The endonuclease activity further supports our previous findings of PALF activity at a 5′ overhang.

The Nuclease Activity of PALF Is Not Affected by Ku or XRCC4-DNA Ligase IV

Previous data have demonstrated binding of PALF to XRCC4 via the FHA domain of PALF and binding to Ku through a region in PALF between the FHA domain and the two CYR (poly [ADP-ribose] binding zinc finger) motifs. We wanted to investigate the possibility that the XRCC4-DNA ligase IV complex or Ku can enhance or block the exonuclease or endonuclease activity of PALF. As above, a single-stranded poly T substrate labeled at the 5′ end was used in incubations with PALF for 1 h with or without purified XRCC4-ligase IV or Ku. The basal activity of PALF generates a combination of endonuclease products and 3′ exonuclease products (Fig. 5, lane 2). XRCC4-DNA ligase IV, after being phosphorylated by CK2, was run alone to rule out the possibility of contamination (Fig. 5, lane 3). XRCC4-DNA ligase IV without phosphorylation was also used as a negative control (Fig. 5, lane 4). The addition of XRCC4-ligase IV did not produce a significant change over basal PALF activity (Fig. 5, lane 2 versus lane 5). A combination of Ku and XRCC4-ligase IV also did not change the product profile (data now shown). Finally, a combination of PALF, XRCC4-ligase IV, and DNA-PKcs failed to have any effect relative to PALF alone (Fig. 5, lane 2 versus lane 6). This is noteworthy, given that both Ku and XRCC4-ligase IV are known to bind to PALF and that DNA-PKcs is suggested to interact with PALF according to proteome analysis (1). Therefore, the interactions do not affect the nuclease activity of PALF.

FIGURE 5.

Effect of Ku, XRCC4-DNA Ligase IV, and DNA-PKcs on PALF nuclease activity. In specified reactions, 50 nm 5′-labeled single-stranded DNA substrate (JG169) was incubated with 125 nm PALF, 126 nm DNA-PKcs, 0.5 mm ATP, 0.5 μm YM8/9, 100 nm Ku, and 75 nm XRCC4-DNA ligase IV in a 10-μl reaction for 60 min at 37 °C. After incubation, reactions were stopped and analyzed by 12% denaturing PAGE.

PALF Is Able to Cooperate with Other NHEJ Factors to Promote Ligation in Vitro and in Vivo

Even though the core NHEJ factors did not stimulate the nuclease activity of PALF or permit it to open hairpins, we were interested in whether PALF could cooperate with other NHEJ factors to stimulate ligation in an in vitro assay. In particular, we wondered if the nuclease activities of PALF could substitute for those of Artemis in resecting overhangs.

We tested this by using an oligonucleotide substrate (SL11/JG258) with four nucleotides of microhomology that had six additional A nts attached to the 3′ end that would require nucleolytic resection before the 4 nts of microhomology could be utilized for ligation. Successful ligation would result in the substrate ligated to another identical molecule in a head-to-tail fashion. When the DNA substrate (Fig. 6A, lane 1) is incubated with XRCC4-ligase IV complex and Ku, it results in a small basal amount of ligation versus substrate alone (Fig. 6A, lane 2 versus lane 1). In the absence of known NHEJ factors, when PALF alone is added to the reaction (Fig. 6A, lane 3), no ligation products are formed. However when PALF, Ku, and XRCC4-ligase IV are all present, ligation products are formed (Fig. 6A, lane 4). PALF is able to significantly increase the amount of product formed versus the basal level (Fig. 6A, lane 4 versus lane 2). The addition of DNA-PKcs to the reaction hinders the ligation process (Fig. 6A, lane 5). It is thought that DNA-PKcs must dissociate before ligation of DNA ends can occur (8, 14, 15). This process is thought to be facilitated by autophosphorylation of DNA-PKcs. However, the precise mechanism is not well understood. In vitro, this process may be too slow in our ligation reactions and hence may inhibit the overall ligation efficiency. The ligation product was subsequently cut out from the gel, cloned into a TA cloning vector, and then sequenced (see “Experimental Procedures”). We found that all ten of the joined product molecules had the six As resected, leaving only the four Cs at the 3′ overhang (Fig. 6C). This allows for efficient joining because of the microhomology between the 4 Cs on the top strand at the 3′ terminus of one duplex substrate and the 4 Gs of the bottom strand of a second duplex substrate. This is also consistent with the results above showing that the PALF nuclease activity terminates its cleavage 3 to 4 nucleotides away from the junction between the overhang and the double-stranded portion of a substrate.

FIGURE 6.

PALF can cooperate with Ku and XRCC4-DNA ligase IV in double-strand DNA end ligation. A, in specified reactions, 50 nm 5′-labeled double-stranded DNA substrate (SL11/JG258) was incubated with 125 nm PALF, 126 nm DNA-PKcs, 0.5 mm ATP, and 0.5 μm YM8/9 for 30 min at 37 °C and followed by the addition of 100 nm Ku and 75 nm XRCC4-DNA ligase IV for 30 min at 37 °C. After incubation, reactions were stopped and analyzed by 8% denaturing PAGE. Positions of the dimerized and trimerized DNA duplex products from the monomeric ligations were determined on the basis of duplex DNA markers not shown on the gel (also see sequencing results in C, which confirm the dimer junctions). B, in specified reactions, 50 nm 5′-labeled double-stranded DNA substrate (SL11/JG258), was incubated with 75 nm Artemis or 125 nm PALF, 126 nm DNA-PKcs, 0.5 mm ATP, and 0.5 μm YM8/9 for 30 min at 37 °C and followed by the addition of 100 nm Ku and 75 nm XRCC4-DNA ligase IV for 30 min at 37 °C. After incubation, reactions were stopped and analyzed by 8% denaturing PAGE. C, dimer products from A, lane 4, were cut out of the gel, extracted, PCR-amplified, TA-cloned, and sequenced. The junctional sequences are shown. PALF removed the AAAAAA (in italics) from each 3′ overhang, thus allowing the 3′-CCCC overhang of one duplex substrate to anneal to the 3′-GGGG on another duplex substrate molecule. These proceeded to ligation by XRCC4-DNA ligase IV to yield the dimer product.

We also compared the efficiency of ligation of PALF versus that of Artemis in an in vitro assay. Using the same substrate (SL11/JG258), we added Ku and XRCC4-DNA ligase IV to Artemis or PALF. Artemis is able to resect the ends in a similar manner as PALF, resulting in ligation using terminal microhomology (Fig. 6B, lane 1). Under the same conditions, PALF is also able to ligate the substrate with a similar efficiency as Artemis (Fig. 6B, lane 3, 12%, versus lane 1, 16%, and supplemental Fig. 4, lane 4 versus lane 1). In some assays, we also added NHEJ factors, XLF and polymerase μ, and observed indistinguishable results (supplemental Fig. 4).

In vivo, we find that knockdown of PALF using siRNA reduces rejoining of two incompatible I-SceI-generated DNA ends by 50% (18) (Fig. 7). This is consistent with recent studies of others that also suggest a role for PALF in NHEJ (19).

FIGURE 7.

siRNA directed against PALF mRNA reduces NHEJ in vivo. A, assay for NHEJ of DSBs in vivo. Two I-SceI sites located in the reverse direction to produce incompatible ends in the substrate DNA are shown as arrowheads. CMV, cytomegalovirus promoter/enhancer; HSV-TK, herpes simplex virus-thymidine kinase; pA, poly(A) signal. Ligation of two broken DNA ends generated by I-SceI digestion results in deletion of the HSV-TK open reading frame and leads to production of a transcript that enables translation of EGFP instead of HSV-TK protein (for details, see Ref. 18). B, Western blot analysis of suppression for PALF expression by siRNA in H1299dA3–1#1 cells. C, PALF is required for end joining of I-SceI-induced double strand breaks in H1299dA3–1#1 cells.

DISCUSSION

PALF Is Both a Single-stranded DNA 3′ Exonuclease and a Single-stranded DNA Endonuclease

The Yasui laboratory (1–3) was the first to demonstrate that PALF (also called APLF) has nuclease activity. Here we confirm that PALF has 3′ exonuclease activity on single-stranded DNA.

We have also found that PALF has endonuclease activity on single-stranded DNA. This activity is capable of cleaving at all sites of a single strand except for the last few nucleotides at the 5′ end. In addition, this activity is able to cleave at all positions of 5′ overhangs of duplex DNA except for the 5′-most few nucleotides of the overhang. Finally, this activity cleaves at all positions of 3′ overhangs except for the approximately four nucleotides 3′ to the boundary of the overhang with the duplex DNA (designated the “no cleavage zone” in Fig. 8).

FIGURE 8.

Summary of exo- and single-stranded endonuclease activities of PALF. PALF has known 3′ exonuclease activity. Here we describe the single-strand DNA endonuclease activity of PALF, which can act at any position within a single-stranded region except within approximately 4 nts of the junction with double-stranded DNA (designated no cleavage zone). The diagonal slash marks on the DNA substrate diagram represent PALF cleavages.

Unification of the PALF Nuclease Activities

We believe that the double-stranded DNA endonuclease activity described previously might be accounted for by the broader single-stranded DNA endonuclease activity described here. The transient breathing of a double-stranded DNA end into single-stranded flaps, which is substantial (20), could serve as a single-stranded substrate for this single-stranded DNA endonuclease activity of PALF. Given the action of PALF on all single-stranded DNA, the endonuclease of PALF has a broader range of substrates than appreciated previously, thereby increasing its potential importance within the cell.

In Vitro and in Vivo Function of PALF with Other NHEJ Proteins

We did not see any stimulation or modification of PALF 3′ exonuclease and single-stranded DNA endonuclease activity by Ku, DNA-PKcs, or XRCC4-DNA ligase IV. Although studies indicate that Ku and XRCC4-DNA ligase IV each bind to PALF (1, 3), these interactions do not appear to affect PALF enzymatically.

Importantly, we find that in an in vitro system consisting of PALF, Ku, and XRCC4-DNA ligase IV is indeed able to join incompatible DNA ends as efficiently as a system consisting of Artemis-DNA-PKcs, Ku, and XRCC4-DNA ligase IV. Specifically, PALF is able to resect the incompatible portion of an overhang to a point at which XRCC4-DNA ligase IV is able to support efficient ligation. Interestingly, at the DNA sequence level, we did not observe any ligation products other than those with the four junctional nucleotides that provide the maximal terminal microhomology. (This does not preclude the possibility that some junctions do contain only three nucleotides and make up a small minority of the products formed.) Use of maximal microhomology could occur by iterative removal of the As by the 3′ exonuclease activity of PALF or by the endonuclease PALF activity. We favor the latter possibility because we did not see a more diverse set of products.

Could PALF and Artemis both function as nucleases in NHEJ? This appears likely, on the basis of our in vivo siRNA knockdown of PALF (Fig. 7). Additionally, PALF suppression results in cells that are sensitive to methyl methane sulphonate (1) as well as sensitive to double-strand break agents.³ Artemis (in complex with DNA-PKcs) may be more efficient in cleaving long overhangs in an endonucleolytic manner (9), whereas PALF has an active 3′ exonuclease activity. Artemis alone (without DNA-PKcs) has a weak single-stranded endonuclease activity (10), and PALF appears to be substantially stronger in this activity. Therefore, in several ways, the nucleolytic activities of Artemis and PALF are complementary to one another.