Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.     

DDB2, DDB1A and DET1 exhibit complex interactions during Arabidopsis development

Damaged DNA-binding proteins 1 and 2 (DDB1 and DDB2) are subunits of the damaged DNA-binding protein complex (DDB). DDB1 is also found in the same complex as DE-ETIOLATED 1 (DET1), a negative regulator of light-mediated responses in plants. Arabidopsis has two DDB1 homologs, DDB1A and DDB1B. ddb1a single mutants have no visible phenotype while ddb1b mutants are lethal. We have identified a partial loss-of-function allele of DDB2. To understand the genetic interaction among DDB2, DDB1A, and DET1 during Arabidopsis light signaling, we generated single, double, and triple mutants. det1 ddb2 partially enhances the short hypocotyl and suppresses the high anthocyanin content of dark-grown det1 and suppresses the low chlorophyll content, early flowering time (days), and small rosette diameter of light-grown det1. No significant differences were observed between det1 ddb1a and det1 ddb1a ddb2 in rosette diameter, dark hypocotyl length, and anthocyanin content, suggesting that these are DDB1A-dependent phenotypes. In contrast, det1 ddb1a ddb2 showed higher chlorophyll content and later flowering time than det1 ddb1a, indicating that these are DDB1A-independent phenotypes. We propose that the DDB1A-dependent phenotypes indicate a competition between DDB2- and DET1-containing complexes for available DDB1A, while, for DDB1A-independent phenotypes, DDB1B is able to fulfill this role.     

DDB1-DDB2 (xeroderma pigmentosum group E) protein complex recognizes a cyclobutane pyrimidine dimer, mismatches, apurinic/apyrimidinic sites, and compound lesions in DNA

The DDB protein complex, comprising the subunits DDB1 and DDB2, binds tightly to UV light-irradiated DNA. Mutations in DDB2 are responsible for xeroderma pigmentosum group E, a disorder with defects in nucleotide excision repair of DNA. Both subunits are also components of a complex involved in ubiquitin-mediated proteolysis. Cellular defects in DDB2 disable repair of the major UV radiation photoproduct in DNA, a cyclobutane pyrimidine dimer, but no significant direct binding of DDB to this photoproduct in DNA has ever been demonstrated. Thus, it has been uncertain how DDB could play a specific role in DNA repair of such damage. We investigated DDB function using highly purified proteins. Co-purified DDB1-DDB2 or DDB reconstituted with individual DDB1 and DDB2 subunits binds to damaged DNA as a ternary complex. We found that DDB can indeed recognize a cyclobutane pyrimidine dimer in DNA with an affinity (K(app)a) 6-fold higher than that of nondamaged DNA. The DDB1-DDB2 complex also bound with high specificity to a UV radiation-induced (6-4) photoproduct and to an apurinic site in DNA. Unexpectedly, DDB also bound avidly to DNA containing a 2- or 3-bp mismatch (and does not bind well to DNA containing larger mismatches). These data indicate that DDB does not detect lesions per se. It instead recognizes other structural features of damaged DNA, acting as a sensor that probes DNA for a subset of conformational changes. Lesions recognized may include those arising when translesion polymerases such as POLH incorporate bases across from DNA lesions caused by UV radiation.     

SUMOylation of damaged DNA-binding protein DDB2

Damaged DNA-binding protein (DDB) is a heterodimer composed of two subunits, p127 and p48, which have been designated DDB1 and DDB2, respectively. DDB2 recognizes and binds to UV-damaged DNA during nucleotide excision repair. Here, we demonstrated that DDB2 was SUMOylated in a UV-dependent manner, and its major SUMO E3 ligase was PIASy as determined by RNA interference-mediated knockdown. The UV-induced physical interaction between DDB2 and PIASy supported this notion. PIASy knockdown reduced the removal of cyclobutane pyrimidine dimers (CPDs) from total genomic DNA, but did not affect that of 6-4 pyrimidine pyrimidone photoproducts (6-4PPs). Thus, DDB2 plays an indispensable role in CPD repair, but not in 6-4PP repair, which is consistent with the observation that DDB2 was SUMOylated by PIASy. These results suggest that the SUMOylation of DDB2 facilitates CPD repair.