After FRAP analyses, the data separated into those for cells with and without mCherry fluorescence. One of the most common environmental genotoxic stressors is ultraviolet (UV) light, which induces characteristic dipyrimidinic DNA photolesions, such as cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). Interfering with replication and transcription, these DNA lesions induce mutations, chromosomal aberrations, and cellular apoptosis, whereas such deleterious effects are counteracted by nucleotide excision repair (NER), a highly versatile DNA repair pathway. In humans, hereditary defects in NER are implicated Rabbit Polyclonal to RCL1 in several autosomal recessive disorders, including xeroderma pigmentosum (XP); seven XP-related genes, through as a heterotrimeric complex, consisting of a human ortholog of yeast Rad23 (RAD23A or B) and centrin-2. Biochemical and structural analyses revealed that the XPC complex is capable of binding specifically to DNA damage sites associated with a relatively large distortion of the DNA duplex by interacting with oscillating normal bases4,5,6. The DNA-bound XPC Neuropathiazol recruits the general transcription factor IIH (TFIIH) complex, and the two ATPase/helicase subunits, XPB and XPD, locally unwind double-stranded DNA. Thereafter, a fully opened complex, containing 24-30 nucleotide long single-stranded DNA, is formed together with XPA and replication protein A (RPA), which serves as an essential structural platform for the subsequent dual incision by two endonucleases, ERCC1/XPF and XPG. The NER dual incision reactions with defined DNA substrates have been reconstituted with the Neuropathiazol six purified protein factors (XPC, TFIIH, XPA, RPA, ERCC1/XPF, and XPG)7. The resulting single-stranded gap is then filled by DNA polymerases in a PCNA-dependent manner, followed by rejoining of the DNA strands with DNA ligases (for details of the NER molecular mechanism, see recent reviews in Ref. 8 & 9). Aside from the core part of the NER process, additional protein factors regulate NER regulatory mechanisms of the DNA damage recognition Neuropathiazol process including both XPC and UV-DDB. Results XPC interacts with SUMO-1 and SUMO conjugating enzymes To understand functional regulation of XPC using purified protein factors, and immunoblot analyses identified a major and several minor shifted bands of XPC in the reaction (Fig. 1b). In contrast with CRL4DDB2-mediated ubiquitination20, the level of XPC SUMOylation was only marginally affected by the presence of DNA (Supplementary Fig. S1a). In addition, Neuropathiazol the DNA-binding activity of XPC was not affected significantly by SUMOylation, regardless of the presence or absence of DNA damage (Supplementary Fig. S1b). Taken together, these data suggest that the SUMOylation of XPC is largely independent of its DNA damage recognition activity. Open in a separate window Figure 1 XPC is SUMOylated and NER dual incision assays of recombinant XPC WT and 3KR. The excised oligonucleotides containing a 6-4PP are indicated. (f) Immunoblot analyses of UVC-treated (10?J/m2) cell lines stably expressing FLAG-XPC (WT or 3KR). After incubation for the indicated times, extracts were prepared and subjected to immunoblot analyses (the same blot was probed sequentially with anti-XPC and anti-RAD23B antibodies). Although HA-DDB2 was ectopically expressed in this experiment to enhance the UV-induced XPC ubiquitination, comparable results were also obtained without DDB2 expression. The asterisk indicates putative SUMOylated XPC bands. To test whether the mutations impaired the basal XPC functions as the initiator of NER, the recombinant XPC 3KR protein was tested in cell-free NER assays. When dual incision of 6-4PP by NER was reconstituted with five purified NER.