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, 286 (42), 36368-77

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

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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

Sicong Li et al. J Biol Chem.

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.

Figures

FIGURE 1.
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.
FIGURE 2.
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.
FIGURE 3.
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.
FIGURE 4.
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.
FIGURE 5.
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.
FIGURE 6.
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.
FIGURE 7.
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.
FIGURE 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.

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