Cockayne syndrome group B protein enhances elongation by RNA polymerase II

Cockayne syndrome (CS) is characterized by impaired physical and mental development. Two complementation groups, CSA and CSB, have been identified. Here we report that the CSB gene product enhances elongation by RNA polymerase II. CSB stimulated the rate of elongation on an undamaged template by a factor of about 3. A thymine-thymine cyclobutane dimer located in the template strand is known to be a strong block to transcription. Addition of CSB to the blocked polymerase resulted in addition of one nucleotide to the nascent transcript. Finally, addition of transcription factor IIS is known to cause polymerase blocked at a thymine-thymine cyclobutane dimer to digest its nascent transcript, and CSB counteracted this transcript shortening action of transcription factor IIS. Thus a deficiency in transcription elongation may contribute to the CS phenotype.

The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells.

Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription.

Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.

Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the XPD (ERCC2) repair/transcription gene

The xeroderma pigmentosum group D (XPD) protein has a dual function, both in nucleotide excision repair of DNA damage and in basal transcription. Mutations in the XPD gene can result in three distinct clinical phenotypes, XP, trichothiodystrophy (TTD), and XP with Cockayne syndrome. To determine if the clinical phenotypes of XP and TTD can be attributed to the sites of the mutations, we have identified the mutations in a large group of TTD and XP-D patients. Most sites of mutations differed between XP and TTD, but there are three sites at which the same mutation is found in XP and TTD patients. Since the corresponding patients were all compound heterozygotes with different mutations in the two alleles, the alleles were tested separately in a yeast complementation assay. The mutations which are found in both XP and TTD patients behaved as null alleles, suggesting that the disease phenotype was determined by the other allele. If we eliminate the null mutations, the remaining mutagenic pattern is consistent with the site of the mutation determining the phenotype.

UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II

Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-damaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptionally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DNA damage induction. Here, we investigated the effect of UV irradiation on transcription by using an in vitro transcription system that allowed uncoupling of initiation from elongation events. Nuclear extracts prepared from UV-irradiated or mock-treated normal human and CS cells were assayed for transcription activity on an undamaged β-globin template. Transcription activity in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a strongly reduced activity, whereas transcription activity was fully restored in extracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced transcription activity in extracts coincided with a strong reduction of RNA polymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the form of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely by blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of transcription. CS proteins may be required for this process in a yet unknown way.