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. 2009 Sep 4;325(5945):1240-3.
doi: 10.1126/science.1177321. Epub 2009 Aug 6.

Poly(ADP-ribose)-dependent regulation of DNA repair by the chromatin remodeling enzyme ALC1

Dragana Ahel  1 , Zuzana Horejsí, Nicola Wiechens, Sophie E Polo, Elisa Garcia-Wilson, Ivan Ahel, Helen Flynn, Mark Skehel, Stephen C West, Stephen P Jackson, Tom Owen-Hughes, Simon J Boulton
Affiliations

Poly(ADP-ribose)-dependent regulation of DNA repair by the chromatin remodeling enzyme ALC1

Dragana Ahel et al. Science. .

Abstract

Posttranslational modifications play key roles in regulating chromatin plasticity. Although various chromatin-remodeling enzymes have been described that respond to specific histone modifications, little is known about the role of poly[adenosine 5'-diphosphate (ADP)-ribose] in chromatin remodeling. Here, we identify a chromatin-remodeling enzyme, ALC1 (Amplified in Liver Cancer 1, also known as CHD1L), that interacts with poly(ADP-ribose) and catalyzes PARP1-stimulated nucleosome sliding. Our results define ALC1 as a DNA damage-response protein whose role in this process is sustained by its association with known DNA repair factors and its rapid poly(ADP-ribose)-dependent recruitment to DNA damage sites. Furthermore, we show that depletion or overexpression of ALC1 results in sensitivity to DNA-damaging agents. Collectively, these results provide new insights into the mechanisms by which poly(ADP-ribose) regulates DNA repair.

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Figures

Figure 1
Figure 1
ALC1 is a chromatin remodelling enzyme regulated by PAR (A) Schematic representation of the wild-type and mutant ALC1 proteins. (B) Purified FLAG- tagged ALC1 proteins stained by Coomassie following SDS gel electrophoresis. (C) In vitro analysis of PAR binding by the wild-type and mutant ALC1 proteins. Proteins were dot-blotted onto a nitrocellulose membrane and incubated with 32P-labelled PAR. APLF was used as a positive control. (D) Interactions with poly(ADP-ribose), PARP1 and core histones are Macro domaindependent. PAR, PARP1 and histones were immunoprecipitated from cells expressing wild-type or mutant FLAG-tagged ALC1 proteins. (E) Quantification of ATPase activities measured by a fluorescence-based assay. ATPase activity of ALC1 (2.3 pmol) is stimulated by the wild-type nucleosomes, but not by the mutant nucleosomes assembled with H4(16-19)A. K77R mutation in the Walker A motif completely abolishes ATPase activity of ALC1. The reactions contained 0.1 mM ATP and 20 nM DNA and nucleosomes. (F) ALC1 repositions nucleosomes in an ATP-dependent manner. 1 pmol of histone octamer assembled on the Cy5-labelled 54A18 DNA was incubated with 2.6 pmol of the wild-type or mutant ALC1 proteins in the absence or presence of 1 mM ATP. Nucleosome-sliding activity of ALC1 is abolished by the Walker A K77R mutation, but not by the Macro domain D723A mutation. (G) Stimulation of ALC1 ATPase activity by PARP1 and NAD+. ATP hydrolysis by recombinant ALC1 (2.3 pmol) was monitored in the presence of nucleosomes. The reactions contained 0.1 mM ATP and 20 nM DNA and nucleosomes. NAD+ (0.5mM), PARP1 (0.16 pmol), PARP inhibitor (1 μM) and purified PAR (15 nM) were added where indicated. Where indicated, PARP1, NAD+ and nucleosomes were preincubated for 5 min before the sequential addition of PARP inhibitor and ALC1. Rates of ATP hydrolysis are expressed relative to the activity of ALC1 in the presence of nucleosomes. (H) Stimulation of ALC1 nucleosome repositioning by PARP1 and NAD+. Nucleosome repositioning assays as in F were performed in the presence of PARP1 (0.16 pmol), NAD+ (0.5mM) and PARP inhibitor (1 μM) as indicated. ATP-dependent redistribution of nucleosomes to distal positions on the fragment is stimulated by PARP in the presence of NAD.
Figure 2
Figure 2
ALC1 associates with DNA repair factors in vivo. (A) Identification of ALC1 interacting partners by mass spectrometry. Chromatin extracts of stable FLAG control and FLAG-tagged ALC1 expressing cells were used for anti-FLAG immunoprecipitation. (B) Numbers of unique peptides identified by mass spectrometry are indicated in the table. (C) ALC1 immunoprecipitates from transiently transfected 293T cells contain known DNA repair factors. Interactions are largely dependent on active PAR synthesis. To minimize nucleic-acid-mediated interactions, extracts were treated with benzonase before anti-FLAG immunoprecipitation.
Figure 3
Figure 3
Recruitment of ALC1 to sites of laser-induced DNA damage. (A) Endogenous ALC1 localizes to sites of laser-induced DNA damage. 1 minute after laser damage, U2OS cells were fixed and stained against γH2AX and ALC1. (B) Recruitment of ALC1 to laser-induced DNA breaks requires active PAR synthesis. U2OS cells were transiently transfected with YFP-ALC1 and treated with PARP inhibitor where indicated. 1 minute after laser damage cells were fixed and stained against γH2AX. (C) Kinetics of ALC1 association and dissociation from DNA breaks. Cells transiently expressing YFP-ALC1 were laser micro-irradiated and YFP fluorescence intensities at sites of laser damage were recorded over time. Mathematical modelling of ALC1 association and dissociation at sites of laser damage was carried out as described in Material and Methods. τ: time constant, N: number of cells. Error bars represent standard deviations. (D) Rapid mobilization and transient association of ALC1 with sites of DNA damage. Shown are representative images of YFP-ALC1 recruitment and dissociation from sites of laser-induced DNA damage. (E) Recruitment of ALC1 to DNA damage sites is Macro domain-dependent, but its efficient dissociation requires active helicase domain. The helicase core fragment of ALC1 (N2 mutant) is not recruited to DNA damage sites and the recruitment of the D723A mutant is dramatically reduced. In contrast, the ATPase dead mutant (K77R) and the Macro domain fragment (C1) show prolonged retention at sites of laser-induced damage. (F) Comparative kinetics of the wild-type and K77R mutant ALC1 recruitment to laser-induced DNA breaks in cells transiently expressing YFP-ALC1 constructs. YFP fluorescence intensities were recorded over time in the laser-damaged region (Damaged) or within an undamaged area in the same nucleus (Undamaged) and normalized against the corresponding fluorescence intensity at the time of laser micro-irradiation. Plotted data are averaged values of a minimum of 10 cells from at least two independent experiments. Error bars represent standard deviations.
Figure 4
Figure 4
Dysregulation of ALC1 expression confers sensitivity to DNA damage. (A) Knockdown efficiency of ALC1 in U2OS stable sh cell lines. (B) Sensitivity of ALC1 deficient cells to DNA damaging agents measured by MTS assay. Plotted data are averaged values from 3 experiments. (C) Over-expression of ALC1 sensitises cells to H2AX phosphorylation. Stable control (Flp-In-FLAG) and FLAG-tagged ALC1 over-expressing cells (Flp-In-ALC1) were either untreated or exposed to 300 μM phleomycin for 1 h, fixed, stained with FITC-γH2AX antibody and analyzed by FACS. Increase in H2AX phosphorylation is abolished by K77R mutation. (D) Assessment of DNA damage in ALC1 over-expressing cells by Comet assay. Stable control (Flp-In-FLAG) and FLAG-tagged ALC1 over-expressing cells (Flp-In-ALC1) were untreated or exposed to the indicated concentrations of phleomycin for 1h, and immediately processed. Increase in DNA breaks is observed in cells overexpressing ALC1 following phleomycin treatment. The difference between untreated FLAG and ALC1 Flp-In cell lines is not statistically significant (P value 0.3941). The differences between phleomycin treated FLAG and ALC1 Flp-In cell lines are statistically significant (P values less than 0.0001). (E) Representative images of control and ALC1 over-expressing cells treated with 0.5 mg/ml phleomycin for 1h and analyzed by Comet assay.
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