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PubMed2000

Transcription-coupled repair of 8-oxoguanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome.

Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences. Defective TCR leads to a mutation frequency at 8-oxoG of 30%-40% compared to the normal 1%-4%. Surprisingly, unrepaired 8-oxoG blocks transcription by RNA polymerase II. These data imply that TCR is required for polymerase release to allow repair and that CS results from defects in TCR of oxidative lesions.

Cell Line↗
PubMed1999

The DNA helicases acting in nucleotide excision repair, XPD, CSB and XPB, are not required for PCNA-dependent repair of abasic sites.

DNA repair of abasic sites is accomplished in mammalian cells by two distinct base excision repair (BER) pathways: a single nucleotide insertion pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway involving a resynthesis patch of 2-10 nucleotides 3' to the lesion. The latter pathway shares some enzymatic components with the nucleotide excision repair (NER) pathway acting on damage induced by ultraviolet light: both pathways are strictly dependent on PCNA and several observations suggest that the polymerization and ligation phases may be carried out by common enzymatic activities (DNA polymerase delta/epsilon and DNA ligase I). Furthermore, it has been postulated that the transcription-NER coupling factor Cockayne syndrome B has a role in BER. We have investigated whether three NER proteins endowed with DNA helicase activities (the xeroderma pigmentosum D and B gene products and the Cockayne syndrome B gene product) may also be involved in repair of natural abasic sites, by using the Chinese hamster ovary mutant cell lines UV5, UV61 and 27-1. No defect of either the PCNA-dependent or the single nucleotide insertion pathways could be observed in UV5, UV61 or 27-1 mutant cell extracts, thus showing that the partial enzymatic overlap between PCNA-dependent BER and NER does not extend to DNA helicase activities.

Animals↗
PubMed1983

CS-US contiguity and CS intensity in conditioning of the rabbit's nictitating membrane response to serial compound stimuli.

The present experiments examined acquisition of the rabbit's nictitating membrane response to serial compound stimuli. Each compound consisted of two distinctive conditioned stimuli (CSA and CSB) that were followed by a shock unconditioned stimulus (US). In Experiment 1, CSA was a highly salient 93-dB tone, and CSB was a moderately salient flashing light. The CSA-US interval was 800 msec, and the CSB-US interval was varied across 400, 600, and 800 msec. In Experiment 2, the flashing light served as CSA, and CSB was either a 73-, a 85-, or a 93-dB tone. The CSA-US interval remained 800 msec, and the CSB-US interval was fixed at 400 msec. The experiments revealed that the CSB-US interval and CSB intensity determined the rate of conditioned response (CR) acquisition to the compound. Yet, CR acquisition to CSB as measured on test trials showed impairments relative to the level of responding in single CS-control conditions. However, the impairment in CR acquisition to CSB was attenuated by increasing CSB-US contiguity, that is, by decreasing the CSB-US interval (Experiment 1) or by increasing CSB intensity (Experiment 2). In Experiment 1, the impairment in acquisition to the light CSB appeared to be primarily a consequence of the tone CSA's greater salience. However, in Experiment 2, impairments in CR acquisition to CSB appeared even when CSB had the combined advantages of CS-US contiguity and great salience relative to CSA. Thus, the results indicated a role for CSA's temporal primacy in determining CR acquisition to the components of a serial compound. The results are discussed in terms of selective-attention, generalization-decrement, and information hypotheses.

Acoustic Stimulation↗
PubMed1993

Effects of extrusion and expelling on the nutritional quality of conventional and Kunitz trypsin inhibitor-free soybeans.

Two chick growth experiments and a precision-fed cockerel digestibility assay were conducted to evaluate the effect of extrusion and expelling on the nutritive value of conventional (CSB) and Kunitz trypsin inhibitor-free (KFSB) soybeans. In the first experiment, performance of chicks fed CSB or KFSB autoclaved at 121 C was similar to that of chicks fed CSB or KFSB extruded at 138 C. The effect of extrusion temperature on protein quality of the soybeans was evaluated in the second experiment. Eleven corn-soybean diets were formulated to contain one of the following: CSB extruded at 104, 121, 138, or 154 C; KFSB extruded at 104, 121, or 138 C; CSB extruded at 121, 138, or 154 C followed by processing through an expeller; and commercial dehulled solvent-extracted soybean meal (SBM). All diets contained 20% crude protein and the same amount of soybean oil and were fed to chicks from 7 to 21 days of age. The CSB extruded at 104 or 121 C and KFSB extruded at 104 C yielded depressed growth and feed efficiency compared with SBM. Performance of chicks on the other treatments was similar to that of chicks fed SBM. Pancreas weight (as a percentage of BW) decreased as extrusion temperature increased, with the response being greater for CSB. Growth performance was greater and pancreas weights were lower for chicks fed KFSB extruded at 104 or 121 C compared with those of chicks fed CSB extruded at the same temperatures. Expelling improved weight gain and feed efficiency when CSB was extruded at 121 C. A 48-h digestibility assay with cecectomized cockerels indicated that digestibility of amino acids in CSB and KFSB increased as extrusion temperature increased and that digestibilities of amino acids in CSB extruded at 104 or 121 C were lower than those in KFSB extruded at the same temperatures. Results of this study indicated that extrusion of CSB at 138 to 154 C or extrusion of KFSB at 121 to 138 C yields protein quality similar to that of SBM.

Amino Acids↗
PubMed1999

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.

4-Nitroquinoline-1-oxide↗
PubMed2001

Disruption of the Cockayne syndrome B gene impairs spontaneous tumorigenesis in cancer-predisposed Ink4a/ARF knockout mice.

Cells isolated from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair, which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS group A (CSA) or group B (CSB) do not exhibit an increased spontaneous or UV-induced cancer rate. In order to investigate the effect of CSB deficiency on spontaneous carcinogenesis, we crossed CSB(-/-) mice with cancer-prone mice lacking the p16(Ink4a)/p19(ARF) tumor suppressor locus. CSB(-/-) mice are sensitive to UV-induced skin cancer but show no increased rate of spontaneous cancer. CSB(-/-) Ink4a/ARF(-/-) mice developed 60% fewer tumors than Ink4a/ARF(-/-) animals and demonstrated a longer tumor-free latency time (260 versus 150 days). Moreover, CSB(-/-) Ink4a/ARF(-/-) mouse embryo fibroblasts (MEFs) exhibited a lower colony formation rate after low-density seeding, a lower rate of H-Ras-induced transformation, slower proliferation, and a lower mRNA synthesis rate than Ink4a/ARF(-/-) MEFs. CSB(-/-) Ink4a/ARF(-/-) MEFs were also more sensitive to UV-induced p53 induction and UV-induced apoptosis than were Ink4a/ARF(-/-) MEFs. In order to investigate whether the apparent antineoplastic effect of CSB gene disruption was caused by sensitization to genotoxin-induced (p53-mediated) apoptosis or by p53-independent sequelae, we also generated p53(-/-) and CSB(-/-) p53(-/-) MEFs. The CSB(-/-) p53(-/-) MEFs demonstrated lower colony formation efficiency, a lower proliferation rate, a lower mRNA synthesis rate, and a higher rate of UV-induced cell death than p53(-/-) MEFs. Collectively, these results indicate that the antineoplastic effect of CSB gene disruption is at least partially p53 independent; it may result from impaired transcription or from apoptosis secondary to environmental or endogenous DNA damage.

Age Factors↗
PubMed1987

Isolation of a cDNA clone for murine catalase and analysis of an acatalasemic mutant.

We have investigated the genetic control of murine catalase expression by analyzing catalase transcription and translation products from the tissues of control (Csa) and acatalasemic (Csb) mouse strains. Csb animals possess nearly normal catalase enzyme activity levels in liver, while displaying approximately 20 and 1% of normal activity levels in kidney and red blood cells, respectively. Immunoblot analyses of catalase in these tissues have revealed reduced levels of immunologically reactive catalase protein in Csb kidney and red blood cells, paralleling the reduction of catalase enzyme activity in these tissues. In order to determine the molecular basis for Csb acatalasemia, we have isolated a cDNA clone for murine catalase and have used this probe to analyze Csa and Csb genomic DNA and catalase mRNA. These studies have revealed: 1) no restriction fragment length polymorphisms between Csa and Csb genomic DNAs; 2) no differences in the levels of Csa and Csb catalase mRNA within a single tissue; and 3) no differences in the sizes of Csa and Csb catalase mRNAs. These observations suggest that the genetic defect that produces the tissue-specific reduction of catalase expression in Csb mice is not due to a marked rearrangement of DNA within the Csb catalase structural gene. Furthermore, the Csb mutation does not act at the level of gene transcription or mRNA stability, but rather at the level of mRNA translation and/or catalase protein turnover.

Acatalasia↗
PubMed2002

Phenotypic consequences of mutations in the conserved motifs of the putative helicase domain of the human Cockayne syndrome group B gene.

Cockayne syndrome (CS) is a human genetic disorder characterized by several neurological and developmental abnormalities. Two genetic complementation groups, CS-A and CS-B, have been identified. The CSB protein belongs to helicase superfamily 2, and to the SWI/SNF family of proteins. The CSB protein is implicated in transcription-coupled repair (TCR), basal transcription and chromatin remodeling. In addition, CS cells undergo UV-induced apoptosis at much lower doses than normal cells. However, the molecular function of the CSB protein in these biological pathways has remained unclear. Evidence indicates that the integrity of the Walker A and B boxes (motifs I and II) are important for CSB function, but the functional significance of the helicase motifs Ia, III--IV has not been previously examined. In this study, single amino acid changes in highly conserved residues of helicase motifs Ia, III, V, VI and a second putative nucleotide-binding motif (NTB) of the CSB protein were generated by site-directed mutagenesis to analyze the genetic function of the CSB protein in survival, RNA synthesis recovery and apoptosis after UV treatment. The survival analysis of these CS-B mutant cell lines was also performed after treatment with the chemical carcinogen, 4-nitroquinoline-1-oxide (4-NQO). The lesions induced by UV light, cyclobutane pyrimidine dimers, are known to be repaired by TCR whereas the lesions induced by 4-NQO are repaired by global genome repair. The results of this study demonstrate that the point mutations in highly conserved residues of helicase motifs Ia, III, V and VI abolished the genetic function of the CSB protein in survival, RNA synthesis recovery and apoptosis after UV treatment. Similarly, the same mutants failed to complement the sensitivity toward 4-NQO. Thus, the integrity of these helicase motifs is important for the biological function of the CSB protein. On the contrary, a point mutation in a C-terminal, second, NTB motif of the CSB protein showed full complementation in the ability to repair damage induced by UV light or 4-NQO, suggesting that this motif is not important for the CSB repair function.

4-Nitroquinoline-1-oxide↗
PubMed2000

Role of the ATPase domain of the Cockayne syndrome group B protein in UV induced apoptosis.

Cockayne syndrome (CS) is a human autosomal recessive disorder characterized by many neurological and developmental abnormalities. CS cells are defective in the transcription coupled repair (TCR) pathway that removes DNA damage from the transcribed strand of active genes. The individuals suffering from CS do not generally develop cancer but show increased neurodegeneration. Two genetic complementation groups (CS-A and CS-B) have been identified. The lack of cancer formation in CS may be due to selective elimination of cells containing DNA damage by a suicidal pathway. In this study, we have evaluated the role of the CSB gene in UV induced apoptosis in human and hamster cells. The hamster cell line UV61 carries a mutation in the homolog of the human CSB gene. We show that both human CS-B and hamster UV61 cells display increased apoptotic response following UV exposure compared with normal cells. The increased sensitivity of UV61 cells to apoptosis is complemented by the transfection of the wild type human CSB gene. In order to determine which functional domain of the CSB gene participates in the apoptotic pathway, we constructed stable cell lines with different CSB domain disruptions. UV61 cells were stably transfected with the human CSB cDNA containing a point mutation in the highly conserved glutamic acid residue in ATPase motif II. This cell line (UV61/ pc3.1-CSBE646Q) showed the same increased apoptosis as the UV61 cells. In contrast, cells containing a deletion in the acidic domain at the N-terminal end of the CSB protein had no effect on apoptosis. This indicates that the integrity of the ATPase domain of CSB protein is critical for preventing the UV induced apoptotic pathway. In primary human CS-B cells, the induction and stabilization of the p53 protein seems to correlate with their increased apoptotic potential. In contrast, no change in the level of either p53 or activation of mdm2 protein by p53 was observed in hamster UV61 cells after UV exposure. This suggests that the CSB dependent apoptotic pathway can occur independently of the transactivation potential of p53 in hamster cells.

Adenosine Triphosphatases↗
PubMed1998

A conserved sequence block in murine and human T cell receptor (TCR) Jalpha region is a composite element that enhances TCR alpha enhancer activity and binds multiple nuclear factors.

A conserved sequence block (CSB) located in a noncoding region of the mouse and human TCR alpha/delta loci, showing six differences over 125 nucleotide positions (95% similar), was subjected to detailed analyses in this study. Transient transfection results showed that the CSB-containing element in conjunction with the TCR alpha enhancer up-regulated the alpha enhancer activity, whereas no enhancer activity was detected when CSB alone was assayed. In vitro occupancy analyses of CSB by nuclear factors reveal the existence of an unexpectedly intricate network of CSB-protein and protein-protein interactions. Lymphoid-specific as well as T-lineage-specific nuclear factors are involved to differentially form CSB-bound complexes in extracts of various tissues and cell lines. Liver was shown to contain factor(s) sequestering thymic CSB-binding factors. Furthermore, the putative binding sites for transcription factors known to be important for lymphoid-lineage development are present in CSB and are targeted by nuclear factors. On the basis of these results, we propose that the CSB element may play a role in shaping the chromatin structure by which the accessibility of TCR alpha/delta loci to the recombinase complex and/or to the transcriptional apparatus can be controlled.

Animals↗
PubMed1997

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.

Cockayne Syndrome↗
PubMed2002

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.

Adenosine Triphosphatases↗
PubMed2000

ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor.

The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

Adenosine Triphosphate↗
PubMed1992

Effect of heating on nutritional quality of conventional and Kunitz trypsin inhibitor-free soybeans.

Five 10-day chick growth experiments and an amino acid digestibility assay were conducted to assess the effect of steam heating on in vivo protein quality of raw, full-fat Kunitz trypsin inhibitor-free soybeans (KFSB) compared with raw, conventional full-fat soybeans (CSB). Protein solubility in .2% KOH was also evaluated as an in vitro test of in vivo protein quality for underprocessed CSB. The CSB and KFSB were autoclaved for 0, 3, 6, 9, 12, 15, 18, or 21 min at 121 C and 124 kPa. The soybeans were then fed to 8- or 9-day-old chicks as the sole source of protein in dextrose and soybean diets containing 23% protein. Growth performance of chicks fed raw KFSB was superior to that of chicks fed raw CSB. Growth performance of chicks fed autoclaved KFSB or CSB increased and pancreas weight (percentage of body weight) decreased as autoclaving time increased. Slightly less autoclaving time was consistently required to achieve maximum chick performance for KFSB compared with CSB. Less autoclaving time was also required to obtain maximum digestibility of amino acids in KFSB compared with CSB. Urease activity of the soybeans decreased as autoclaving time increased, whereas protein solubility in .2% KOH for CSB did not change consistently in response to heating time. The results of the present study indicate that raw KFSB must be heated to obtain maximum protein quality for chicks and that protein solubility in KOH is not a sensitive indicator of underprocessing of CSB soybeans.

Amino Acids↗
PubMed2000

Impact of global genome repair versus transcription-coupled repair on ultraviolet carcinogenesis in hairless mice.

The nucleotide excision repair (NER) system is comprised of two subpathways, i.e., transcription-coupled repair (TCR) and global genome repair (GGR). To establish the relative importance of TCR and GGR for UV effects on the skin, we have used hairless knockout mouse strain lacking either TCR (CSB -/-) or GGR (XPC -/-). In single exposure experiments, we found that CSB -/- mice have a 7-16 times higher susceptibility to sunburn than XPC -/- mice and than heterozygous (+/-) and wild-type (+/+) controls. Exposure to 80 J/m2 UV radiation (i.e., suberythemogenic in CSB -/-) on 10 consecutive days gives rise to epidermal hyperplasia in CSB -/- and XPC -/-, whereas repair-proficient controls do not show epidermal hyperplasia from these exposures. In addition, CSB -/- mice develop marked parakeratosis, whereas XPC -/- mice and controls do not. Under continued exposure to this daily dose, squamous cell carcinomas appear in CSB -/-, XPC -/-, and in the control groups, whereas only in the CSB -/- animals is a fairly high number of benign papillomas also found. The median latency time of squamous cell carcinomas (diameters > or = 1 mm) is 84 days for the XPC -/- mice, 115 days for the CSB -/- mice, and 234-238 days for the heterozygous and wild-type control groups. These results indicate that GGR is more important than TCR in protection against UV-induced carcinomas of the skin but not against other UV effects such as sunburn, epidermal thickening, scaling of the stratum corneum, and development of papillomas. These results also indicate that GGR capacity may serve as a better predictor for skin cancer susceptibility than sensitivity to sunburn. The relative cancer susceptibilities of GGR- and TCR-deficient skin could well depend on the balance between an increased mutation rate and the presence (in CSB -/-) or lack (in XPC -/-) of a compensatory apoptotic response.

Animals↗