Leucine zipper-like domain is required for tumor suppressor ING2-mediated nucleotide excision repair and apoptosis

The plant homodomain (PHD) of ING2 was shown to regulate p53-dependent apoptosis through phosphoinositides signaling. However, the role of a predicted leucine zipper-like (LZL) motif in N-terminus of ING2 is unclear. Here, we show that LZL motif is critical for the proper functions of ING2 in DNA repair, apoptosis and chromatin remodeling after UV irradiation. Deletion of LZL domain also abrogated the association between ING2 and p53, but not between ING2 and p300, suggesting that ING2 modulates p53-dependent chromatin remodeling, apoptosis and DNA repair by functioning as a scaffold protein to mediate the interaction between p53 and p300.     

The chromatin remodeling factor BRG1 stimulates nucleotide excision repair by facilitating recruitment of XPC to sites of DNA damage

BRG1 is a catalytic subunit of the human SWI/SNF-like BAF chromatin remodeling complexes. Recent findings have shown that inactivation of BRG1 sensitizes mammalian cells to various DNA damaging agents, including ultraviolet (UV) and ionizing radiation. However, it is unclear whether BRG1 facilitates nucleotide excision repair (NER). Here we show that re-expression of BRG1 in cells lacking endogenous BRG1 expression stimulates nucleotide excision repair of UV induced DNA damage. Using a micropore UV radiation technique, we demonstrate that recruitment of the DNA damage recognition protein XPC to sites of UV lesions is significantly disrupted when BRG1 is depleted. Chromatin immunoprecipitation of the endogenous DDB2 protein, which is involved in recruiting XPC to UV-induced CPDs (cyclobutane pyrimidine dimers), shows that elevated levels of BRG1 are associated with DDB2 in chromatin in response to UV radiation. Additionally, we detected slow BRG1 accumulation at sites of UV lesions. Our findings suggest that the chromatin remodeling factor BRG1 is recruited to sites of UV lesions to facilitate NER in human chromatin.     

New histone incorporation marks sites of UV repair in human cells

Chromatin organization is compromised during the repair of DNA damage. It remains unknown how and to what extent epigenetic information is preserved in vivo. A central question is whether chromatin reorganization involves recycling of parental histones or new histone incorporation. Here, we devise an approach to follow new histone deposition upon UV irradiation in human cells. We show that new H3.1 histones get incorporated in vivo at repair sites. Remarkably we find that H3.1, which is deposited during S phase, is also incorporated outside of S phase. Histone deposition is dependent on nucleotide excision repair (NER), indicating that it occurs at a postrepair stage. The histone chaperone chromatin assembly factor 1 (CAF-1) is directly involved in the histone deposition process in vivo. We conclude that chromatin restoration after damage cannot rely simply on histone recycling. New histone incorporation at repair sites both challenges epigenetic stability and possibly contributes to damage memory.     

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4^Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4^Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.     

The p38 mitogen-activated protein kinase augments nucleotide excision repair by mediating DDB2 degradation and chromatin relaxation

The p38 MAPK is a family of serine/threonine protein kinases that play important roles in cellular responses to external stress signals, e.g. UV irradiation. To assess the role of p38 MAPK pathway in nucleotide excision repair (NER), the most versatile DNA repair pathway, we determined the efficiency of NER in cells treated with p38 MAPK inhibitor SB203580 and found that p38 MAPK is required for the prompt repair of UV-induced DNA damage CPD. We further investigated the possible mechanism through which p38 MAPK regulates NER and found that p38 MAPK mediates UV-induced histone H3 acetylation and chromatin relaxation. Moreover, p38 MAPK also regulates UV-induced DDB2 ubiquitylation and degradation via phosphorylation of the target protein. Finally, our results showed that p38 MAPK is required for the recruitment of NER factors XPC and TFIIH to UV-induced DNA damage sites. We conclude that p38 MAPK regulates chromatin remodeling as well as DDB2 degradation for facilitating NER factor assembly.     

Tumor suppressor Ing1b facilitates DNA repair and prevents oxidative stress induced cell death

Inhibitor of growth (ING) family of proteins are known to coordinate with histone acetyltransferases and regulate the key events of cell cycle and DNA repair. Previous work from our lab showed that Ing1b regulated the nucleotide excision repair by facilitating histone acetylation and subsequent chromatin relaxation. Further, it was also shown that Ing1b protected the cells from genomic instability induced cell death by promoting ubiquitination of proliferating cell nuclear antigen (PCNA). In the present study we explored the role of Ing1b in the repair of oxidized DNA and prevention of oxidative stress induced genotoxic cell death. Using HCT116 cells we show that Ing1b protein expression is induced by treatment with H₂O₂. Ing1b lacking cells showed decreased ability to repair the oxidized DNA. PCNA monoubiquitination, a critical event of DNA repair was blunted in Ing1b knock down cells and augmented in Ing1b over expressing cells. Moreover, oxidative stress induced cell death was higher in cells lacking Ing1b whereas it was lower in Ing1b over expressing cells. Finally we show that inhibition of histone deacetylases, rescued the Ing1b knock down cells from cytotoxic effects of H₂O₂ treatment.     

The yeast histone chaperone chromatin assembly factor 1 protects against double-strand DNA-damaging agents

The removal of histones from DNA and their subsequent replacement is likely to be necessary for all processes that require access to the DNA sequence in eukaryotic cells. The histone chaperone chromatin assembly factor 1 (CAF-1) mediates histone H3-H4 assembly during DNA replication and nucleotide excision repair in vitro. We have found that budding yeast deleted for the genes encoding CAF-1 are highly sensitive to double-strand DNA-damaging agents. Our genetic analyses indicate that CAF-1 plays a role in both homologous recombination and nonhomologous end-joining pathways and that the function of CAF-1 during double-strand repair is distinct from that of another histone H3-H4 chaperone, anti-silencing function 1 (ASF1). CAF-1 does not protect the genome by assembling it into a damage-resistant chromatin structure, because induction of CAF-1 after DNA damage is sufficient to restore viability. Furthermore, CAF-1 is not required for repair of the DNA per se or for DNA damage checkpoint function. CAF-1-mediated resistance to DNA damage is dependent on the ability of CAF-1 to bind PCNA, indicating that PCNA may recruit CAF-1 to sites of double-strand DNA repair. We propose that CAF-1 has an essential role in assembling chromatin during double-strand-DNA repair.     

Role of p21WAF1 in the cellular response to UV

UV or g irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21WAF1 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21WAF1 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21WAF1 function.