Monoubiquitinated histone H2A destabilizes photolesion-containing nucleosomes with concomitant release of UV-damaged DNA-binding protein E3 ligase

Abstract

How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4B(DDB2). We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4B(DDB2) E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4B(DDB2) from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.

FIGURE 1.

DDB1-CUL4B^DDB2 E3 ligase binds mononucleosomes reconstituted with UV-damaged DNA. A, shown is the experimental strategy for the assembly of nucleosomes used as a binding substrate for the DDB1-CUL4B^DDB2 E3 ligase in pulldown studies. B, mononucleosomes were reconstituted in vitro from the mock or UV-irradiated nucleosome-positioning DNA sequence and human recombinant octamers and analyzed on ethidium bromide-stained agarose gel. C, the reconstituted nucleosomes with undamaged DNA (nucleosomes) or UV-damaged DNA (UV nucleosomes) were incubated ± DDB1-CUL4B^DDB2, and the DNA was pulled down with streptavidin-bound Dynabeads. The supernatants (20%) and pulled-down beads were boiled and run on a gradient SDS-PAGE. The separated proteins were probed with the DDB1, DDB2, and H2A antibodies. Samples: supernatant (Sup) and pulled-down material (P.D.) containing 4 pmol of mononucleosomes without E3 (lanes 1 and 2), 4 pmol of mononucleosomes and 2 pmol of E3 (lanes 3 and 4), and 4 pmol of mononucleosomes and 4 pmol of E3 (lanes 5 and 6). D, shown is the presence of core histones in a complex ±DDB1-CUL4B^DDB2 after pulling down the UV-damaged nucleosomes, confirmed by Coomassie staining of the gel. The input of the binding assay (16 pmol of the E3 and 36 pmol of histone octamers) and pulled-down UV-damaged nucleosomes (32 pmol) were separated as in C. The strong staining overlapping with histone H4 is a nonspecific staining of the beads (marked with stars). E, recombinant proteins, P. tridactylis CPD photolyase (P.t. CPD phr) and A. thaliana 6-4 photolyase (A.t. 6-4 phr) expressed in E. coli, were purified with chitin beads and Q-Sepharose and visualized with Coomassie staining. Samples: chitin bead eluted fraction (lane 1), Q-Sepharose flow-through (lane 2), and concentrated Q-Sepharose elution (5 μg) (lane 3). F, UV-DNA was photoreactivated (PHR: photo-repaired) with 6-4PP or CPD photolyase to generate the nucleosome-positioning DNA sequences with CPD or 6-4PP photoproducts, respectively. The indicated amounts of DNA (1 μg of 177-bp nucleosome-positioning DNA sequence is equivalent to 8 pmol) photoreactivated with one or both photolyases were spotted on nitrocellulose membranes and probed with CPD or 6-4PP antibodies. Samples of ±UV-irradiated DNA were included as controls. G, shown is binding of DDB1-CUL4B^DDB2 (2 pmol for the left panel or 4 pmol for the right panel) to ±UV-irradiated and ±photoreactivated DNA (4 pmol) (left panel) and mononucleosomes assembled with CPD or 6-4PP-DNA (8 pmol) (right panel), tested as in C.

FIGURE 2.

DDB1-CUL4B^DDB2 E3 ligase targets tagged nucleosomal H2A and H3 for ubiquitination in a photolesion binding-dependent manner. Free Myc-H2A (A) or HA-H3 (B) was used as a substrate for DDB1-CUL4B^DDB2 E3 ligase (4 pmol). Each ubiquitination assay was performed ±FLAG-ubiquitin (FLAG-Ub) in triplicate. The total reaction was separated on 15% SDS-PAGE. Modified histone was detected with uH2A, FLAG, and Myc in A and with H3, FLAG, and HA antibodies in B. C, free Myc-H2A and nucleosomal Myc-H2A (nucleosomes or UV-nucleosomes) in C or free HA-H3 and nucleosomal HA-H3 (UV- nucleosomes) in D were used as a substrate for DDB1-CUL4B^DDB2 E3 ligase (8 pmol). Membrane strips were probed with DDB1 and Myc (C) or HA (D) antibodies.

FIGURE 3.

DDB1-CUL4B^DDB2 ligase activity in targeting H2A mutants. A, ubiquitination of a free Myc-H2A histone was assayed in the presence of unmodified ubiquitin (FLAG-Ub) or mutated ubiquitin: Ub-K48 only (single lysine residue, Lys-48) or Ub-no lysine (all lysine residues mutated). Unmodified and modified H2A was detected with Myc antibody. B, wild type or H2A mutants (H2A K119R, H2A K120R, and H2A K119R/K120R) were used as a substrate for DDB1-CUL4B^DDB2 E3 ligase, and products were detected as in A. C, shown is expression of FLAG-His-tagged wild type (WT) or H2A mutants (H2A K119R, H2A K120R, and H2A K119R/K120R) in the chromatin soluble fraction of the stable isogenic 293 cell lines 30 min after ±UV irradiation of 40 J/m². Unmodified and modified histones were detected with FLAG antibody at the position of 20 and 27 kDa, respectively. The other bands, detected in the chromatin fractions above monoubiquitinated-H2A, presumably represent a different type of modification that occurred endogenously or after damage.

FIGURE 4.

Monoubiquitinated H2A Lys-119 and Lys-120 facilitate disassembly of a UV-damaged nucleosome. A, shown is the experimental design of the nucleosomal disassembly assay. Immobilized UV-damaged nucleosomes (wild type H2A or H2A K119R/K120R) were used as a substrate for the ubiquitination assay ± DDB1-CUL4B^DDB2 E3. Unless indicated the ubiquitination reaction was supplemented with ATP. After the reaction was terminated, the supernatant and beads (DNA-bound fraction) were divided. Fractions from sample 1 were loaded on the gel as a total yield of unmodified and modified histone per ubiquitin reaction in vitro. Beads from the sample, run in a parallel reaction (sample 2), were washed with buffer using increasing amounts of NaCl and boiled with sample buffer. Samples were run on 4–20% gradient SDS-PAGE, and the membrane was probed with Myc antibody to detect modified and unmodified H2A (B) and H3 antibody to detect modified and unmodified H3 (C).

FIGURE 5.

Histone mutant H2A K119R/K120R prevents dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. Samples were prepared as outlined in Fig. 4A with a modified ubiquitination assay; E3 was a component in all reactions, and ATP was supplemented as indicated (±ATP). Samples were run on a 4–20% gradient SDS-PAGE gel after which the membrane strips were probed with DDB1 and DDB2 antibodies (A) and the whole membrane was probed with DDB2 antibody (B).

FIGURE 6.

Proposed model for the role of ubiquitinated H2A Lys-119 and Lys-120 in UV-damaged nucleosomes in the initiation of NER. DDB2 binds to a 6-4PP or CPD photolesion and positions the DDB1-CUL4B^DDB2 E3 ligase over the damaged nucleosome. The E3 ligase facilitates the transfer of activated ubiquitin from UbcH5 (E2) to the core histone H2A Lys-119/Lys-120 and to DDB2 itself. Monoubiquitination of the H2A Lys-119/Lys-120 leads to dissociation of H2A, presumably as (H2A-H2B) dimers, and destabilization of the nucleosome. This destabilization facilitates release of polyubiquitinated DDB2, freeing space around the lesion for loading of the NER preincision complex. Nucleosomes in which these lysines are replaced with arginines (i.e. H2A K119R/K120R) are resistant to such structural changes; thus, the polyubiquitinated DDB2 is not disassociated from the damaged nucleosome, leading instead to the release of the DDB1-CUL4-RBX1 E3 subcomplex. Note that Andrews et al. (76) described an equilibrium constant for the transition between 2(H2A/H2B)-(H3-H4)₂-DNA and 2(H2A/H2B) + DNA-(H3-H4)₂ nucleosomal structural states, confirming a function of tetrasomes ((H3-H4)₂-DNA), after eviction of the 2(H2A/H2B).