Abstract
Blunted melanocortin 1 receptor (MC1R) signaling promotes melanocyte genomic instability in part by attenuating cAMP-mediated DNA repair responses, particularly nucleotide excision repair (NER), which recognizes and clears mutagenic photodamage. cAMP-enhanced NER is mediated by interactions between the ataxia telangiectasia-mutated and Rad3-related (ATR) and xeroderma pigmentosum complementation group A (XPA) proteins. We now report a critical role for sirtuin 1 (SIRT1) in regulating ATR-mediated phosphorylation of XPA. SIRT1 deacetylates XPA at residues Lys-63, Lys-67, and Lys-215 to promote interactions with ATR. Mutant XPA containing acetylation mimetics at residues Lys-63, Lys-67, and Lys-215 exhibit blunted UV-dependent ATR-XPA interactions even in the presence of cAMP signals. ATR-mediated phosphorylation of XPA on Ser-196 enhances cAMP-mediated optimization of NER and is promoted by SIRT1-mediated deacetylation of XPA on Lys-63, Lys-67, and Lys-215. Interference with ATR-mediated XPA phosphorylation at Ser-196 by persistent acetylation of XPA at Lys-63, Lys-67, and Lys-215 delays repair of UV-induced DNA damage and attenuates cAMP-enhanced NER. Our study identifies a regulatory ATR-SIRT1-XPA axis in cAMP-mediated regulation melanocyte genomic stability, involving SIRT1-mediated deacetylation (Lys-63, Lys-67, and Lys-215) and ATR-dependent phosphorylation (Ser-196) post-translational modifications of the core NER factor XPA.
Highlights
Our study supports a model of cAMP–DNA repair enhancement that utilizes functional cross-talk between deacetylation and phosphorylation and identifies a regulatory ATR–sirtuin 1 (SIRT1)–xeroderma pigmentosum complementation group A (XPA) axis in the nucleotide excision repair (NER) pathway
Because our earlier work documented that cAMP-enhanced NER depended on ATR–XPA interactions [17], we considered whether other post-translational modifications in ATR or XPA might regulate this pathway
Since deacetylation of XPA by SIRT1 was reported to be an important regulator of NER following UV radiation [38], we considered whether SIRT1 is involved in cAMP-enhanced NER
Summary
Our previous work documented a cAMP-dependent pathway that regulates NER pertinent to MC1R signaling in melanocytes. The specificity of the assay was confirmed by showing lack of nonspecific staining in the negative controls, displayed by the omission of either CPD or SIRT1 antibody alone (Fig. S1) Together, these results suggest that ATR promotes localization of SIRT1 to sites of UV-induced DNA damage. We noted robust interaction by co-immunoprecipitation (co-IP) between SIRT1 and XPA in UV-irradiated A375 melanoma cells Their interaction was dramatically attenuated by the addition of either a kinase-dead ATR or ATR–P2445L (Fig. 1C). We conclude that UV-dependent SIRT1-mediated XPA deacetylation is ATR-dependent, because expression of ATR–KD or ATR–P2445L ablated the SIRT1-dependent deacetylation of XPA following UV-induced DNA damage (Fig. 1D) These data collectively support a UV-induced ATR– SIRT–XPA axis, wherein ATR function is needed for SIRT1 localization to sites of photodamage, association with XPA, and deacetylation of XPA following UV.
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