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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.
Highlights
The compaction of DNA into nucleosomes interferes with DNA repair
In previous studies we demonstrated that mutations in DDB2 alter the co-localization of the DDB1-CUL4DDB2 ligases at the site of UV lesions and the monoubiquitination of the surrounding H2A [20], resulting in the partial impairment of global genome repair in the cells of xeroderma pigmentosum group E patients compared with repair-proficient cells
DDB1-CUL4BDDB2 E3 Ligase Binds to cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) Lesions in Mononucleosomes—To define the role of human recombinant DDB1-CUL4BDDB2 E3 ligase in the ubiquitination of core histones, we developed an in vitro system with the photoproducts incorporated into a reconstituted mononucleosome in which one of the human core histones is replaced with tagged proteins (Fig. 1A)
Summary
The compaction of DNA into nucleosomes interferes with DNA repair. Results: Monoubiquitination of core histone H2A destabilizes nucleosomes containing UV-damaged DNA. Repair proteins responsible for recognizing the helix distortion in the nucleosome direct chromatin modulators to the site of the DNA lesion to facilitate the removal of core histones [12]. The direct link between DDB1-CUL4DDB2 and the UV-damage-dependent ubiquitination of other core histones has been confirmed in vitro and in vivo [21], suggesting that this type of modification could facilitate NER in the compact chromatin of the non-transcribed DNA. We sought to examine the consequences of damage-dependent ubiquitination by DDB1-CUL4BDDB2 E3 ligase on nucleosome dynamics utilizing mononucleosomes assembled with human recombinant-tagged histone octamers and nucleosome-positioning DNA containing CPD or 6-4PP photolesions. We show for the first time a link between the ubiquitination of H2A lysines 119 and 120, the destabilization of the UVdamaged nucleosome, resulting from the eviction of the H2A, and the dissociation of polyubiquitinated DDB2 from the nucleosome
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