To understand the mechanism of nucleotide excision repair (NER), one of the major human DNA repair pathways, we have set up a DNA repair system in which a linear damaged DNA substrate is immobilized by its terminus. By isolating functionally active intermediate complexes, our data dissect the ordered arrival and displacement of NER factors in the progress of the dual incision step. We describe (i) the role of ATP in remodelling the NER-initiating complex of XPC/TFIIH/damaged DNA as a prerequisite for the recruitment of the next NER factors; (ii) the coordination between damage removal and DNA resynthesis and the release of XPC-HR23B, TFIIH and XPA upon arrival of XPG and XPF-ERCC1, respectively; (iii) how RPA remains associated with the excised DNA initiating the assembly of resynthesis factors such as PCNA; (iv) the recycling of XPC-HR23B, TFIIH and XPA in the NER; and the shuttling of TFIIH between NER and transcription. Thus, our ®ndings de®ne multiple functions of NER factors to explain the molecular basis of human NER disorders.

Retinoic acid receptor ␥ (RAR␥) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RAR␥1 and serines 66 and 68 in RAR␥2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7⅐cyclin H⅐MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RAR␥ is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RAR␥ phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RAR␥ transcriptional activity in a response gene-dependent manner.The pleiotropic effects of retinoids are transduced by two nuclear receptor families, the retinoic acid receptors (RARs) 1 and the retinoid X receptors (RXRs), that are ligand-dependent transregulators belonging to the nuclear receptor superfamily (1-4). RARs are activated by all-trans and 9-cis retinoic acid, whereas RXRs are activated by 9-cis retinoic acid only. There are three RAR (␣, ␤, and ␥) and three RXR (␣, ␤, and ␥) isotypes, and for each isotype there are at least two main isoforms that differ in their N-terminal region (1, 5, 6).As do other members of the nuclear receptor superfamily, RARs and RXRs exhibit a conserved modular structure with six variably conserved regions (A to F) (Fig. 1) (1, 5). The Nterminal A/B region of RARs contains a ligand-independent transcriptional activation function, AF-1 (7, 8). Although the B regions of the three RAR isotypes are moderately conserved, their A regions are unrelated and differ for each isoform of a given RAR isotype (5). The highly conserved C region encompasses the central DNA binding domain. The function of region F, if any, is unknown. Region E is more complex, as it contains the ligand binding domain, a dimerization interface, and the ligand-dependent transcriptional activation/repression domain AF-2 (1, 9). The activity of AF-2 is entirely dependent on the integrity of a conserved sequence referred to as the AF-2 AD core, located in ␣-helix 12 at the C-terminal end of the ligand binding domain. Ligand binding induces a major conformational change that includes helix 12 and creates a new surface for coactivator binding while corepressors are released, thus resulting in a transcriptional-competent nuclear receptor relayed to the transcriptional machinery and the chromatin template (1, 10 -12). The AF-2 and AF-1 activities synergize with each other in a response element-and promoter context-dependent manner (1,8,13).RARs and RXRs are phosphoproteins (14 -16), and their phosphorylation involves several kinases. RAR␣ can be phosphorylated in its AF-1-containing B region by the cyclin-dependent kinase cdk7 (14), which together with MAT1 and cyclin H forms the CAK complex that is found either free or as a component of the g...