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...

To address the biochemical mechanisms underlying the coordination between the various proteins required for nucleotide excision repair (NER), we employed the immobilized template system. Using either wild-type or mutated recombinant proteins, we identified the factors involved in the NER process and showed the sequential comings and goings of these factors to the immobilized damaged DNA. Firstly, we found that PCNA and RF-C arrival requires XPF 5 0 incision. Moreover, the positioning of RF-C is facilitated by RPA and induces XPF release. Concomitantly, XPG leads to PCNA recruitment and stabilization. Our data strongly suggest that this interaction with XPG protects PCNA and Pold from the effect of inhibitors such as p21. XPG and RPA are released as soon as Pold is recruited by the RF-C/PCNA complex. Finally, a ligation system composed of FEN1 and Ligase I can be recruited to fully restore the DNA. In addition, using XP or trichothiodystrophy patient-derived cell extracts, we were able to diagnose the biochemical defect that may prove to be important for therapeutic purposes.

Elongating RNA polymerase II blocked by DNA damage in the transcribed DNA strand is thought to initiate the transcription-coupled repair process. The objective of this study is to better understand the sequence of events that occurs during repair from the time RNA polymerase II first encounters the lesion. This study establishes that an immobilized DNA template containing a unique cisplatin lesion can serve as an in vitro substrate for both transcription and DNA repair. RNA polymerase II is quantitatively stalled at the cisplatin lesion during transcription and can be released from the template, along with the nascent transcript, in an ATPdependent manner. RNA polymerase II stalled at a lesion and containing a dephosphorylated repetitive carboxyl-terminal domain (CTD) appears to be more sensitive toward release. However, a dephosphorylated CTD can become readily phosphorylated in front of the lesion by CTD kinases in the presence of ATP. The observation that RNA polymerase II and transcript release occurs in a TFIIH-deficient repair extract but not in a reconstituted repair system demonstrates that disassembly of the elongation complex can occur independently of the repair process and vice versa. Indeed, the presence of RNA polymerase II at the lesion does not prevent dual incision from occurring. Finally, we also propose that the Cockayne's syndrome B protein factor, believed to be the mammalian transcription repair coupling factor, is neither involved in transcript release nor required for dual incision in the presence of lesionstalled RNA polymerase II in vitro. More likely, it prevents RNA polymerase from backing up when it encounters the lesion. The ability to transcribe and repair the same damaged DNA molecule fixed on beads, along with the fact that the reaction conditions can be freely altered, provides a powerful tool to study the fate of RNA polymerase II blocked on the cisplatin lesion.

BackgroundBone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding.Methodology/Principal FindingsUsing in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor β (PDGFR-β) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts.Conclusions/SignificanceWe conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-β signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

Phosphorylation of the estrogen receptor alpha (ERalpha) N-terminal transcription activation function AF1 at serine 118 (S118) modulates its activity. We show here that human ERalpha is phosphorylated by the TFIIH cyclin-dependent kinase in a ligand-dependent manner. Furthermore, the efficient phosphorylation of S118 requires a ligand-regulated interaction of TFIIH with AF2, the activation function located in the ligand binding domain (LBD) of ERalpha. This interaction involves (1) the integrity of helix 12 of the LBD/AF2 and (2) p62 and XPD, two subunits of the core TFIIH. These findings are suggestive of a novel mechanism by which nuclear receptor activity can be regulated by ligand-dependent recruitment of modifying activities, such as kinases.

No abstract

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla(TM)). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA') fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA', was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA'-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.