HuCOP1 contributes to the regulation of DNA repair in keratinocytes.

We have previously demonstrated that the E3 ligase Human Constitutive Photomorphogenic Protein (huCOP1) is expressed in human keratinocytes and negatively regulates p53. The MutS homolog 2 (MSH2) protein plays a central role in DNA MMR mechanism and is implicated in the cellular response to anticancer agents, such as cisplatin. Our aim was to clarify whether huCOP1 plays a role in DNA MMR by affecting MSH2 protein level in human keratinocytes. To define the role of huCOP1 in DNA mismatch repair, we determined whether huCOP1 affects MSH2 abundance. MSH2 protein level was detected by immunocytochemical staining using a keratinocyte cell line in which the expression level of huCOP1 was stably decreased (siCOP1). To investigate whether huCOP1 silencing influences cisplatin-induced cell death, control and siCOP1 keratinocyte cells were treated with increasing concentrations of cisplatin and cell viability was recorded after 48 and 96 h. Stable silencing of huCOP1 in human keratinocytes resulted in a reduced level of MSH2 protein. huCOP1 silencing also sensitized keratinocytes to the interstrand crosslinking inducer cisplatin. Our results indicate that decreased huCOP1 correlates with lower MSH2 levels. These protein level changes lead to increased sensitivity toward cisplatin treatment, implicating that huCOP1 plays a positive role in maintaining genome integrity in human keratinocytes.

The RAD30 cancer susceptibility gene.

The human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in RAD30, which encodes the novel lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and increased mutagenesis following UV irradiation. In cell-free extracts of XPV lymphoblasts, functional DNA polymerase eta is required for the complete replication of a double-stranded plasmid containing either a single (6-4) photoproduct or a cyclobutane pyrimidine dime(CPD), the major mutagenic UV-induced lesion. In cultured XPV cells, replication arrest activates downstream signalling pathways, leading to hyperphosphorylation of the 34-kDa subunit of the trimeric single-stranded DNA-binding protein, RPA (replication protein A). Many of the RAD30 mutations identified in XPV cells result in truncation and inactivation of DNA polymerase eta. To examine whether polymorphisms in the RAD30 gene that result in altered polymerase eta function, rather than enzyme inactivation, might contribute to individual susceptibility to skin cancer, methods to screen for sequence changes in the RAD30 gene in human genomic DNAhave been developed.

A single (6-4) photoproduct inhibits plasmid DNA replication in xeroderma pigmentosum variant cell extracts.

The human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in the human RAD30 gene, which encodes the lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and increased mutagenesis following UV-irradiation. Using extracts of an XPV lymphoblast cell line (GM2449C) that has a truncating mutation in the RAD30 gene, we investigated the effect of a (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD), at a unique -TT- site on either the leading or lagging strand, on plasmid DNA replication. Compared to normal cell extracts, XPV cell extracts have a reduced capacity to carry out complete replication of DNA containing either a (6-4) photoproduct or a CPD on the leading strand, whereas there is little difference between the two cell extracts in replication of DNA containing a lesion on the lagging strand. Inhibition of replication in the presence of a (6-4) photoproduct is attributed to arrest of nascent DNA strand synthesis at the lesion site; in XPV cell extracts, the proportion of arrested products is increased compared to that of normal cell extracts. These results are consistent with a requirement for functional DNA polymerase eta in the replication of a double-stranded plasmid containing either a (6-4) photoproduct or a CPD, on the leading but not the lagging strand.

UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein.

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

In vitro replication and mutagenesis of a novel reversion vector with selective DNA damage in the supF gene.

The ability to detect the most common type of UV-induced mutation, the C to T transition, at the previously characterized hotspot at position 99 of the supF gene has been demonstrated in a selectively irradiated reversion vector, pLS189(Rev). The supF region was amplified, irradiated with 500J/m(2) UVC or unirradiated, and ligated into the pLS189(Rev) plasmid. A portion of ligated product plasmid containing the irradiated fragment was sensitive to nicking by T4 endonuclease V, indicating the presence of the most common type of UV-induced damage, the pyrimidine dimer. Plasmid containing the irradiated or unirradiated supF gene was replicated completely in cellular extracts from either HeLa or XP-A cells in vitro. Plasmid containing the irradiated supF gene showed an inhibition of total replication to a level similar to those of previous studies with plasmid molecules exposed in their entirety to 40J/m(2). Replication of selectively irradiated plasmid resulted in an average reversion frequency of 0.071% in the two extracts; a 42-fold increase over the average spontaneous reversion frequency of unirradiated plasmid. The reversion frequencies were not significantly different between extracts prepared from HeLa and XP-A cells, indicating that neither the repair status of the cell lines nor the XPA protein itself affect the frequency of C to T transitions at position 99 of the supF gene in plasmid replicated in vitro. These data indicate that the plasmid pSL189(Rev), containing the selectively UV-irradiated supF gene, is a useful and sensitive tool to study mutagenesis at a specific site. This approach may be applicable to the investigation of other environmental DNA-damaging agents, by allowing the target gene to be selectively damaged while maintaining the ability of the plasmid to replicate completely. Such a system, amenable to biochemical manipulation, may be very valuable in elucidating the function of novel proteins in the process of mutagenesis.

Expression of ATM in ataxia telangiectasia fibroblasts rescues defects in DNA double-strand break repair in nuclear extracts.

Ataxia telangiectasia (A-T) is a human genetic disorder characterized by progressive cerebellar degeneration, hypersensitivity to ionizing radiation (IR), immunodeficiency, and high cancer risk. At the cellular level, IR sensitivity and increased frequency of spontaneous and IR-induced chromosomal breakage and rearrangements are the hallmarks of A-T. The ATM gene, mutated in this syndrome, has been cloned and codes for a protein sharing homology with DNA-PKcs, a protein kinase involved in DNA double-strand break (DSB) repair and DNA damage responses. The characteristics of the A-T cellular phenotypes and ATM gene suggest that ATM may play a role similar to that of DNA-PKcs in DSB repair and that there is a primary DNA repair defect in A-T cells. In the current study, the function of ATM in DNA DSB repair was evaluated in an in vitro system using two plasmids, carrying either an EcoRI-induced DSB within the lacZalpha gene or various endonuclease-induced DSB in the SupF suppressor tRNA gene. We found that the DSB repair efficiency in A-T nuclear extracts was comparable to, if not higher than, that in normal nuclear extracts. However, the repair fidelity in A-T nuclear extracts was decreased when repairing DSB with short 5' and 3' overhangs (<4 base pairs (bp)) or blunt ends, but not 5' 4-bp overhangs. Sequencing of the mutant plasmids revealed that deletions involving 1-6 nucleotide microhomologies were the major class of mutations in both A-T and normal extracts. However, the size of the deletions in plasmids from A-T nuclear extracts was larger than that from normal nuclear extracts. Expression of the ATM protein in A-T cells corrected the defect in DSB repair in A-T nuclear extracts. These results suggest that ATM plays a role in maintaining genomic stability by preventing the repair of DSB from an error-prone pathway.

Regulation of human airway mucins by acrolein and inflammatory mediators.

Bronchitis, asthma, and cystic fibrosis, marked by inflammation and mucus hypersecretion, can be caused or exacerbated by airway pathogens or irritants including acrolein, an aldehyde present in tobacco smoke. To determine whether acrolein and inflammatory mediators alter mucin gene expression, steady-state mRNA levels of two airway mucins, MUC5AC and MUC5B, were measured (by RT-PCR) in human lung carcinoma cells (NCI-H292). MUC5AC mRNA levels increased after >/=0.01 nM acrolein, 10 microM prostaglandin E2 or 15-hydroxyeicosatetraenoic acid, 1.0 nM tumor necrosis factor-alpha (TNF-alpha), or 10 nM phorbol 12-myristate 13-acetate (a protein kinase C activator). In contrast, MUC5B mRNA levels, although easily detected, were unaffected by these agonists, suggesting that irritants and associated inflammatory mediators increase mucin biosynthesis by inducing MUC5AC message levels, whereas MUC5B is constitutively expressed. When transcription was inhibited, TNF-alpha exposure increased MUC5AC message half-life compared with control level, suggesting that transcript stabilization is a major mechanism controlling increased MUC5AC message levels. Together, these findings imply that irritants like acrolein can directly and indirectly (via inflammatory mediators) increase airway mucin transcripts in epithelial cells.

The morphological and spectral phenotype of apoptosis in HeLa cells varies following exposure to UV-C and the addition of inhibitors of ICE and CPP32.

Numerous extra- and intracellular factors, including UV radiation, can initiate a programme of cell death by apoptosis. While apoptosis is commonly defined morphologically, the relationships between morphology and molecular events are not well established. To investigate these relationships in HeLa cells, eight morphometric criteria for cell proliferation and damage and 10 criteria for apoptotic phenotype were examined using light microscopy, and corroborated by ultrastructure and spectral imaging. They were identified (1) during a time course after irradiation with 0, 10 or 30 J/m2 UV-C; (2) after separation of apoptotic from normal cells on a Percoll gradient; and (3) after irradiation with UV-C plus perturbation of the apoptotic pathway by treatment with inhibitors of two caspases, ICE and CPP32. The number of cells in apoptosis increased in a dose-dependent manner after UV-C treatment. Centrifugation of irradiated cells on a Percoll gradient increased the collection of apoptotic cells tenfold. The stereotypical apoptotic phenotype, in which cells have deep cytoplasmic blebbing and highly condensed DNA, comprised only a few percent of all apoptosis, and was rarely seen in groups receiving caspase inhibitors. The most common apoptotic phenotype was a rounded cell with large spherical nucleolus and associated DNA. After treatment with UV-C plus inhibitors the apoptotic index was decreased by about 30% compared to UV-C radiation alone. These apoptotic cells had dark spherical cytoplasm with small blebs, greatly increased numbers of cytoplasmic ribosomes, abundant nucleolar material with a large separate granular component, and chromatin condensed at the nuclear membrane. Using the technique of spectral imaging, it was found that the spectrum obtained from the granular component of the nucleolus, which was elevated in apoptotic cells treated with UV-C plus inhibitors, was similar to the dense accumulation of ribosomes in the apoptotic cytoplasm. The data indicate that spectral imaging may be a useful tool for identifying and characterizing variations in the apoptotic process, and that the caspase inhibitors used here do not completely abolish UV-C induced apoptosis, but rather alter its incidence and progression.

Complete replication of plasmid DNA containing a single UV-induced lesion in human cell extracts.

To investigate the effect of the major UV-induced lesions on SV40 origin-dependent DNA replication and mutagenesis in a mammalian cell extract, double-stranded plasmids containing a single cis,syn-cyclobutane dimer or a pyrimidine-pyrimidone (6-4) photoproduct at a unique TT sequence have been constructed. These plasmids have been used as templates in DNA replication-competent extracts from human HeLa cells. Plasmids containing a single pyrimidine cyclobutane dimer on the potential lagging strand for DNA replication are replicated with an efficiency approximately equal to that of an unmodified plasmid. A small decrease in replication efficiency of approximately 20% was observed when the lesion was located on the potential leading strand for DNA replication. In both orientations, DpnI-resistant, replicated closed circular plasmid DNA was sensitive to nicking by the pyrimidine dimer-specific enzyme, T4 endonuclease V, indicating that complete replication of the damaged plasmid occurs in vitro. In contrast, a (6-4) photoproduct, within the same site and sequence context on the lagging strand for DNA synthesis, inhibits replication in vitro by an average of approximately 50%, indicating that the mammalian replication complex responds differently to the two major UV-induced lesions during DNA replication in vitro. Analysis of the DpnI-resistant, replicated DNA for mutations targeted to the lesion site indicates that neither of these lesions resulted in significant mutagenesis. UV-induced lesions at TT sites may therefore be poorly mutagenic under these conditions for DNA replication in human cell extracts in vitro.

Analysis of mutations induced by replication of UV-damaged plasmid DNA in HeLa cell extracts.

We have used an SV40-based shuttle vector, pZ189, to investigate the capacity of HeLa cell extracts to reproduce the in vivo process of mutation fixation. We showed previously that when UV-irradiated pZ189 is replicated in these extracts, bypass of UV photoproducts occurs, resulting in base substitution mutations in the supF gene of the vector. Here we report the DNA sequence characterization of a collection of 60 of these UV-induced mutants. Most of the mutations observed are single or tandem double base substitutions at dipyrimidine sites; of these, approximately 90% are G:C-->A:T transitions. Mutations are observed predominantly at a few sites, in particular at positions 155 and 156 in the supF sequence. No dramatic differences in the mutation spectrum were observed when the orientation of the supF gene was reversed with respect to the SV40 origin of replication, suggesting that mutation fixation occurs similarly on both the leading and the lagging strands for DNA replication. Generally, the mutational hot spots observed in vitro are at the same sites as those observed when UV-irradiated pZ189 was passaged in human or monkey cells in culture. Thus, it appears that the replication and mutagenesis of UV-damaged templates in HeLa cell extracts accurately reflects these processes in the intact cell.

UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein.

We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus.

Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts.

We have used in vitro DNA replication systems from human HeLa cells and monkey CV-1 cells to replicate a UV-damaged simian virus 40-based shuttle vector plasmid, pZ189. We found that replication of the plasmid was inhibited in a UV fluence-dependent manner, but even at UV fluences which caused damage to essentially all of the plasmid molecules some molecules became completely replicated. This replication was accompanied by an increase (up to 15-fold) in the frequency of mutations detected in the supF gene of the plasmid. These mutations were predominantly G:C-->A:T transitions similar to those observed in vivo. Treatment of the UV-irradiated plasmid DNA with Escherichia coli photolyase to reverse pyrimidine cyclobutane dimers (the predominant UV-induced photoproduct) before replication prevented the UV-induced inhibition of replication and reduced the frequency of mutations in supF to background levels. Therefore, the presence of pyrimidine cyclobutane dimers in the plasmid template appears to be responsible for both inhibition of replication and mutation induction. Further analysis of the replication of the UV-damaged plasmid revealed that closed circular replication products were sensitive to T4 endonuclease V (a pyrimidine cyclobutane dimer-specific endonuclease) and that this sensitivity was abolished by treatment of the replicated DNA with E. coli photolyase after replication but before T4 endonuclease treatment. These results demonstrate that these closed circular replication products contain pyrimidine cyclobutane dimers. Density labeling experiments revealed that the majority of plasmid DNA synthesized in vitro in the presence of bromodeoxyuridine triphosphate was hybrid density whether or not the plasmid was treated with UV radiation before replication; therefore, replication of UV-damaged templates appears to occur by the normal semiconservative mechanism. All of these data suggest that replication of UV-damaged templates occurs in vitro as it does in vivo and that this replication results in mutation fixation.

HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro.

To determine whether cellular replication factors can influence the fidelity of DNA replication, the effect of HeLa cell single-stranded DNA-binding protein (SSB) on the accuracy of DNA replication by HeLa cell DNA polymerase alpha has been examined. An in vitro gap-filling assay, in which the single-stranded gap contains the supF target gene, was used to measure mutagenesis. Addition of SSB to the in vitro DNA synthesis reaction increased the accuracy of DNA polymerase alpha by 2- to 8-fold. Analysis of the products of DNA synthesis indicated that SSB reduces pausing by the polymerase at specific sites in the single-stranded supF template. Sequence analysis of the types of errors resulting from synthesis in the absence or presence of SSB reveals that, while the errors are primarily base substitutions under both conditions, SSB reduces the number of errors found at 3 hotspots in the supF gene. Thus, a cellular replication factor (SSB) can influence the fidelity of a mammalian DNA polymerase in vitro, suggesting that the high accuracy of cellular DNA replication may be determined in part by the interaction between replication factors, DNA polymerase and the DNA template in the replication complex.

Simian virus 40 (SV40) small t antigen inhibits SV40 DNA replication in vitro.

We describe a biochemical function of simian virus 40 small t antigen, the inhibition of simian virus 40 large T antigen-mediated viral DNA replication in an in vitro replication system. Our results suggest that in this system, small t antigen prevents protein phosphatase 2A-mediated activation of large T antigen.

DNA polymerase alpha from HeLa cells synthesizes DNA with high fidelity in a reconstituted replication system.

To determine the contribution that DNA polymerase alpha makes to the overall DNA replication fidelity in mammalian systems, we measured the fidelity of replication of the SV40-based shuttle vector, pZ189, in a reconstituted in vitro DNA replication system which contained purified HeLa DNA polymerase alpha (in addition to single-stranded DNA binding protein, topoisomerase II, DNA ligase, 5'----3' exonuclease, ribonuclease H, and SV40 T-antigen). We found that DNA polymerase alpha is highly accurate when carrying out bidirectional replication in this system. This high fidelity of replication by DNA polymerase alpha in the reconstituted replication system contrasts with a relatively low fidelity of gap-filling DNA synthesis on the same target gene by purified HeLa cell DNA polymerase alpha in the absence of other replication factors. The fidelity of DNA replication by DNA polymerase alpha, although relatively high in the reconstituted system, is about 4-fold lower than DNA replication in a crude HeLa cell extract which contains additional replication factors including DNA polymerase delta. These results demonstrate that DNA polymerase alpha has the capacity to replicate DNA with high fidelity when carrying out semiconservative DNA replication in a minimal reconstituted replication system, but additional cellular factors not present in the reconstituted system may contribute to the higher replication fidelity of the crude system.

Effect of glucagon on hepatic phenylalanine hydroxylase in vivo.

Moderate doses of glucagon (20 micrograms/kg I.V.) are sufficient to stimulate rat hepatic phenylalanine hydroxylase in vivo. In addition, the stimulation of the tetrahydrobiopterin-dependent phenylalanine hydroxylase activity in livers of animals fed on a high-protein diet has been correlated with an elevated phosphate content. The tetrahydrobiopterin-dependent hydroxylase activity in these animals can be further elevated by glucagon-stimulated phosphorylation. These results indicate that physiological changes in glucagon concentration modulate rat liver phenylalanine hydroxylase activity in vivo. The current understanding of the role of phosphorylation in regulating human phenylalanine hydroxylase is also considered.