Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.

Restoration of UV-inhibited transcription requires removal of transcription-blocking DNA lesions by transcription-coupled repair (TCR). In mammals, TCR is dependent on CSA and CSB proteins; however, their functions are largely unknown. Here, we analyzed the composition of UV-stalled transcription elongation complexes from human cells. We show that CSB and CSA display differential roles in recruitment of TCR-specific factors and that assembly for TCR occurs without disruption of the UV-stalled RNA polymerase II (RNAPIIo). CSB fulfills a key role as a coupling factor to attract histone acetyltransferase p300, nucleotide excision repair (NER) proteins, and CSA-DDB1 E3-ubiquitin ligase complex with the COP9 signalosome. CSA is dispensable for attraction of NER proteins to lesion-stalled RNAPIIo, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1, and TFIIS. These results give insight into the nature and order of molecular events that take place during TCR in the context of chromosomal DNA.

DNA polymerase carries out translesion synthesis past UV photoproducts and is deficient in xeroderma pigmentosum (XP) variants. We report that pol is mostly localized uniformly in the nucleus but is associated with replication foci during S phase. Following treatment of cells with UV irradiation or carcinogens, it accumulates at replication foci stalled at DNA damage. The C-terminal third of pol is not required for polymerase activity. However, the C-terminal 70 aa are needed for nuclear localization and a further 50 aa for relocalization into foci. Pol truncations lacking these domains fail to correct the defects in XP-variant cells. Furthermore, we have identified mutations in two XP variant patients that leave the polymerase motifs intact but cause loss of the localization domains.

The encounter of elongating RNA polymerase II (RNAPIIo) with DNA lesions has severe consequences for the cell as this event provides a strong signal for P53-dependent apoptosis and cell cycle arrest. To counteract prolonged blockage of transcription, the cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER), a specialized subpathway of nucleotide excision repair (NER). Exposure of mice to UVB light or chemicals has elucidated that TC-NER is a critical survival pathway protecting against acute toxic and long-term effects (cancer) of genotoxic exposure. Deficiency in TC-NER is associated with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). Recent data suggest that CSA and CSB play differential roles in mammalian TC-NER: CSB as a repair coupling factor to attract NER proteins, chromatin remodellers and the CSA-E3-ubiquitin ligase complex to the stalled RNAPIIo. CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1 and TFIIS. The emerging picture of TC-NER is complex: repair of transcription-blocking lesions occurs without displacement of the DNA damage-stalled RNAPIIo, and requires at least two essential assembly factors (CSA and CSB), the core NER factors (except for XPC-RAD23B), and TC-NER specific factors. These and yet unidentified proteins will accomplish not only efficient repair of transcription-blocking lesions, but are also likely to contribute to DNA damage signalling events.

No abstract

Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-α, HP1-β, and HP1-γ are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage.

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.

Cells from patients with Cockayne syndrome (CS) are hypersensitive to UV-irradiation but have an apparently normal ability to remove pyrimidine dimers from the genome overall. We have measured the repair of pyrimidine dimers in defined DNA sequences in three normal and two CS cell strains. When compared to a nontranscribed locus, transcriptionally active genes were preferentially repaired in all three normal cell strains. There was no significant variation in levels of repair between various normal individuals or between two constitutively expressed genes, indicating that preferential repair may be a consistent feature of constitutively expressed genes in human cells. Neither CS strain, from independent complementation groups, was able to repair transcriptionally active DNA with a similar rate and to the same extent as normal cells, indicating that the genetic defect in CS lies in the pathway for repair of transcriptionally active DNA. These results have implications for understanding the pleiotropic clinical effects associated with disorders having defects in the repair of DNA damage. In particular, neurodegeneration appears to be associated with the loss of preferential repair of active genes and is not simply correlated with reduced levels of overall repair.Cockayne syndrome (CS) is an autosomal recessive disorder characterized by growth retardation, skeletal and retinal abnormalities, progressive neurological degeneration, and severe photosensitivity. At the cellular level, CS is characterized by an increased sensitivity to the killing effects of UV-irradiation and, in response to UV-irradiation, CS cells show elevated levels of sister chromatid exchanges and reduced host-cell reactivation of irradiated viruses and are hypermutable (1). These responses indicate that CS cells are defective in their ability to repair DNA damage. Analysis of cultured CS cells indicated an apparently normal capacity for removing UV-induced DNA damage. The rate of removal of T4 endonuclease-sensitive sites, levels of repair replication, postreplication repair, incision frequencies, and ligation of repair patches after UV-irradiation fell within the normal range (2). This is in contrast to the UV-sensitive disorder xeroderma pigmentosum (XP) in which UV sensitivity has been correlated with the inability to excise UV-induced DNA damage or to seal daughter-strand gaps left after DNA synthesis on a damaged template (1).Despite the apparently normal ability of CS cells to remove DNA damage, CS cells were unable to restore normal levels of RNA and DNA synthesis after UV-irradiation, a defect also seen in excision-defective XP cells (3). After low fluences of UV-irradiation, normal cells showed a depression of RNA synthesis but recovery to unirradiated levels was rapid and occurred before the removal of the bulk of pyrimidine dimers. As pyrimidine dimers are blocks to the transcription machinery (4) and as recovery of RNA synthesis occurred before the removal of these blocks in the genome overall, Mayne and Lehmann (3) propose...