Role In Transcription-coupled Repair Research Articles

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

Genome-wide transcription-coupled repair in Escherichia coli is mediated by the Mfd translocase.

We used high-throughput sequencing of short, cyclobutane pyrimidine dimer-containing ssDNA oligos generated during repair of UV-induced damage to study that process at both mechanistic and systemic levels in Escherichia coli Numerous important insights on DNA repair were obtained, bringing clarity to the respective roles of UvrD helicase and Mfd translocase in repair of UV-induced damage. Mechanistically, experiments showed that the predominant role of UvrD in vivo is to unwind the excised 13-mer from dsDNA and that mutation of uvrD results in remarkable protection of that oligo from exonuclease activity as it remains hybridized to the dsDNA. Genome-wide analysis of the transcribed strand/nontranscribed strand (TS/NTS) repair ratio demonstrated that deletion of mfd globally shifts the distribution of TS/NTS ratios downward by a factor of about 2 on average for the most highly transcribed genes. Even for the least transcribed genes, Mfd played a role in preferential repair of the transcribed strand. On the other hand, mutation of uvrD, if anything, slightly pushed the distribution of TS/NTS ratios to higher ratios. These results indicate that Mfd is the transcription repair-coupling factor whereas UvrD plays a role in excision repair by aiding the catalytic turnover of excision repair proteins.

A role for SUMO in nucleotide excision repair

The two Siz/PIAS SUMO E3 ligases Siz1 and Siz2 are responsible for the vast majority of sumoylation in Saccharomyces cerevisiae. We found that siz1Δ siz2Δ mutants are sensitive to ultra-violet (UV) light. Epistasis analysis showed that the SIZ genes act in the nucleotide excision repair (NER) pathway, and suggested that they participate both in global genome repair (GGR) and in the Rpb9-dependent subpathway of transcription-coupled repair (TCR), but have minimal role in Rad26-dependent TCR. Quantitative analysis of NER at the single-nucleotide level showed that siz1Δ siz2Δ is deficient in repair of both the transcribed and non-transcribed strands of the DNA. These experiments confirmed that the SIZ genes participate in GGR. Their role in TCR remains unclear. It has been reported previously that mutants deficient for the SUMO conjugating enzyme Ubc9 contain reduced levels of Rad4, the yeast homolog of human XPC. However, our experiments do not support the conclusion that SUMO conjugation affects Rad4 levels. We found that several factors that participate in NER are sumoylated, including Rad4, Rad16, Rad7, Rad1, Rad10, Ssl2, Rad3, and Rpb4. Although Rad16 was heavily sumoylated, elimination of the major SUMO attachment sites in Rad16 had no detectable effect on UV resistance or removal of DNA lesions. SUMO attachment to most of these NER factors was significantly increased by DNA damage. Furthermore, SUMO-modified Rad4 accumulated in NER mutants that block the pathway downstream of Rad4, suggesting that SUMO becomes attached to Rad4 at a specific point during its functional cycle. Collectively, these results suggest that SIZ-dependent sumoylation may modulate the activity of multiple proteins to promote efficient NER.

Diverse Roles of RNA Polymerase II-associated Factor 1 Complex in Different Subpathways of Nucleotide Excision Repair

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two pathways of nucleotide excision repair (NER). In Saccharomyces cerevisiae, Rad26 is important but not absolutely required for TCR. Rpb4, a nonessential RNA polymerase II (Pol II) subunit that forms a subcomplex with Rpb7, and the Spt4-Spt5 complex, a transcription elongation factor, have been shown to suppress Rad26-independent TCR. The Pol II-associated factor 1 complex (Paf1C) has been shown to function in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that Paf1C plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooperating with Spt4-Spt5. We propose a model that, in the absence of Rad26, a lesion is "locked" in the active center of a Pol II elongation complex, which is stabilized by the coordinated interactions of Rpb4-Rpb7, Spt4-Spt5, and Paf1C with each other and with the core Pol II. We also found that Paf1C facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of histone H2B lysine 123 by Bre1, which in turn permits di- and trimethylation of histone H3 lysine 79 by Dot1. To our best knowledge, among the NER-modulating factors documented so far, Paf1C appears to have the most diverse functions in different NER pathways or subpathways.

Evidence That the Histone Methyltransferase Dot1 Mediates Global Genomic Repair by Methylating Histone H3 on Lysine 79

Global genomic repair (GGR) and transcription coupled repair (TCR) are two pathways of nucleotide excision repair (NER) that differ in the damage recognition step. How NER factors, especially GGR factors, access DNA damage in the chromatin of eukaryotic cells has been poorly understood. Dot1, a histone methyltransferase required for methylation of histone H3 lysine 79 (H3K79), has been shown to confer yeast cells with resistance to DNA-damaging agents and play a role in activation of DNA damage checkpoints. Here, we show that Dot1 and H3K79 methylation are required for GGR in both nucleosomal core regions and internucleosomal linker DNA, but play no role in TCR. H3K79 trimethylation contributes to but is not absolutely required for GGR, and lower levels of H3K79 methylation (mono- and dimethylation) also promote GGR. Our results also indicate that the roles of Dot1 and H3K79 methylation in GGR are not achieved by either activating DNA damage checkpoints or regulating the expression of the GGR-specific factor Rad16. Rather, the methylated H3K79 may serve as a docking site for the GGR machinery on the chromatin. Our studies identified a novel GGR-specific NER factor and unveiled the critical link between a covalent histone modification and GGR.

Impaired Repair of Ionizing Radiation-Induced DNA Damage in Cockayne Syndrome Cells

Cockayne syndrome (CS) cells are defective in transcription-coupled repair (TCR) and sensitive to oxidizing agents, including ionizing radiation. We examined the hypothesis that TCR plays a role in ionizing radiation-induced oxidative DNA damage repair or alternatively that CS plays a role in transcription elongation after irradiation. Irradiation with doses up to 100 Gy did not inhibit RNA polymerase II-dependent transcription in normal and CS-B fibroblasts. In contrast, RNA polymerase I-dependent transcription was severely inhibited at 5 Gy in normal cells, indicating different mechanisms of transcription response to X rays. The frequency of radiation-induced base damage was 2 × 10(-7) lesions/base/Gy, implying that 150 Gy is required to induce one lesion/30-kb transcription unit; no TCR of X-ray-induced base damage in the p53 gene was observed. Therefore, it is highly unlikely that defective TCR underlies the sensitivity of CS to ionizing radiation. Overall genome repair levels of radiation-induced DNA damage measured by repair replication were significantly reduced in CS-A and CS-B cells. Taken together, the results do not provide evidence for a key role of TCR in repair of radiation-induced oxidative damages in human cells; rather, impaired repair of oxidative lesions throughout the genome may contribute to the CS phenotype.

Differential requirement for the ATPase domain of the Cockayne syndrome group B gene in the processing of UV-induced DNA damage and 8-oxoguanine lesions in human cells.

Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by UV sensitivity, developmental abnormalities and premature aging. The cellular and molecular phenotypes of CS include increased sensitivity to oxidative and UV-induced DNA lesions. The CSB protein is thought to play a pivotal role in transcription-coupled repair and CS-B cells are defective in the repair of the transcribed strand of active genes, both after exposure to UV and in the presence of oxidative DNA lesions. A previous study has indicated that a conserved helicase ATPase motif II residue is essential for the function of the CSB protein in responding to UV-induced DNA damage in a hamster cell line. Due to the limitations in studying a complex human disorder in another species, this study introduced the site-directed mutation of the ATPase motif II in the human CSB gene in an isogenic human cell line. The CSB mutant allele was tested for genetic complementation of UV-sensitive phenotypes in the human CS-B cell line CS1AN.S3.G2. In addition, the incision of an 8-oxoguanine lesion by extracts of the CS-B cell lines stably transfected with the wild-type or ATPase mutant CSB gene has been investigated. The ATPase motif II point mutation (E646Q) abolished the function of the CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery and apoptosis. Interestingly, whole-cell extract prepared from these mutant cells retained wild-type incision activity on an oligonucleotide containing a single 8-oxoguanine lesion, whereas the absence of the CSB gene altogether resulted in reduced incision activity relative to wild-type. These results suggest damage-specific functional requirements for CSB in the repair of UV-induced and oxidative lesions in human cells. The transfection of the mutant or wild-type CSB gene into the CS1AN.S3.G2 cells did not alter the expression of the subset of genes examined by cDNA array analysis.

Excision repair at the level of the nucleotide in the upstream control region, the coding sequence and in the region where transcription terminates of the Saccharomyces cerevisiae MFA2 gene and the role of RAD26

RAD26, the yeast homologue of human CSB, has an essential role in transcription-coupled repair (TCR). We have mapped the requisite of Rad26 for nucleotide excision repair (NER) within the different regions of the yeast Saccharomyces cerevisiae MFA2 gene at nucleotide resolution. Our results show that Rad26 is dispensable for enhanced NER in both the MFA2 upstream promoter, except in the TATA box region, and for enhanced NER in both strands of the active gene at a site close to the transcription termination region. As expected, it is not needed for repair of regions downstream of where transcription terminates. However, it is required for TCR in the transcription initiation and elongation regions. Our data support the hypothesis that Rad26 is required for the interchange between holo-TFIIH and a putative repairosome containing core TFIIH and other NER proteins. Close to the end of transcription, hotspots for the repair of CPDs in both the transcribed strand and the non-transcribed strand occur. This enhanced repair is independent of Rad26. Hence, TFIIH may take a form favourable for forming a repairosome without Rad26 assistance; here the organisation of the DNA during the termination of transcription may facilitate access of a repair complex to enable enhanced repair of both strands.

Recruitment of the putative transcription-repair coupling factor CSB/ERCC6 to RNA polymerase II elongation complexes.

Cockayne's syndrome (CS) is a disease characterized by developmental and growth defects, sunlight sensitivity, and a defect in transcription-coupled nucleotide excision repair. The two principle proteins involved in CS, CSA and CSB/ERCC6, have been hypothesized to bind RNA polymerase II (Pol II) and link transcription to DNA repair. We have tested CSA and CSB in assays designed to determine their role in transcription-coupled repair. Using a unique oligo(dC)-tailed DNA template, we provide biochemical evidence that CSB/ERCC6 interacts with Pol II molecules engaged in ternary complexes containing DNA and nascent RNA. CSB is a DNA-activated ATPase, and hydrolysis of the ATP beta-gamma phosphoanhydride bond is required for the formation of a stable Pol II-CSB-DNA-RNA complex. Unlike CSB, CSA does not directly bind Pol II.

The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex.

Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

Transcription-Coupled DNA Repair

Certain DNA lesions are removed preferentially from the transcribed strands of active genes in bacteria, yeast and mammalian cells. Initially it was thought that only lesions removed by nucleotide excision repair (NER) were subject to this pathway of transcription-coupled repair (TCR), but recent investigations have shown that some lesions caused by reactive oxygen species, that are recognized by glycosylases and are subject to base excision repair (BER), can be preferentially repaired in the transcribed strands of active genes in mammalian cells. We will discuss the pathways and the proteins involved in the repair of different lesions or groups of lesions, produced by environmental agents or endogenous metabolic activities. The victims of the human hereditary diseases, xeroderma pig-mentosum (XP) and Cockayne syndrome (CS) are highly sensitive to DNA lesions induced by sunlight but only the former exhibit predisposition to cancer. While XP patients are generally deficient in NER, those with CS are specifically defective in TCR and are characterized by dwarfism and severe developmental abnormalities. The role of TCR in human health will be discussed with specific consideration of the problems of cancer and early development.