Transcription factor IIH - the protein complex with multiple functions.

Transcription factor IIH (TFIIH) is eukaryotic multi protein complex identified in early 90's. Subsequent years have shown exceptional conservation of its structure from yeast to human. Although initially considered to be exclusively a basal transcription factor responsible for initiation of transcription and transition from initiation to elongation, TFIIH is also important in nucleotide excision repair for opening DNA at the site of a lesion and for recruitment of additional repair factors. Recently it was suggested that intact holocomplex of TFIIH is required for cell cycle regulation. Moreover, mutations in TFIIH subunits lead to three distinct genetic disorders: xeroderma pigmentosum (DNA repair disorder/cancer syndrome), Cockayne syndrome (DNA repair disorder/transcription syndrome/segmental progeria) and trichothiodystrophy (DNA repair disorder/transcription syndrome). This review is focused on the TFIIH structure, its role in transcription, DNA repair and cell cycle regulation and association with some human hereditary disorders.

Involvement of the nucleotide excision repair proteins in the removal of oxidative DNA base damage in mammalian cells.

Oxidative DNA base damage produced primarily by reactive oxygen species is assumed to be the most important endogenous damage. Lack of its repair may contribute to mutagenesis, carcinogenesis and aging. It is supposed that most oxidative DNA base damage is removed by the base excision repair pathway; although it was shown recently that other DNA repair pathways could be involved. This review is focused on the role of nucleotide excision repair (NER) and transcription-coupled repair (TCR) in the removal of oxidative DNA base damage in mammalian cells.

Role of DNA polymerase II in the tolerance of thymine dimers remaining unexcised in UV-irradiated Escherichia coli exposed to pre-UV nutritional stress.

Nutritional stress applied prior to UV-irradiation to E. coli 15 555-7 reduced thymine dimer excision and inhibited post-UV incorporation of thymidine in polB(+) as well as in polB(-) cells. However, the pre-UV-stressed polB(+) cells were significantly more UV-resistant and after UV synthesized larger DNA molecules than the pre-UV-stressed polB(-) cells. The data suggest that DNA polymerase II is involved in the tolerance of unremoved thymine dimers.

Approaches to identification of HNPCC suspected patients in Slovak population.

Patients with hereditary non-polyposis colorectal cancer (HNPCC) have a DNA mismatch repair defect (MMR) in their tumor tissue that results in instability of microsatellite DNA sequences (MSI). Thus, MSI analysis may effectively indicate this form of cancer that should be then proved by analysis of germline mutations in MMR genes. The aim of this study was to identify HNPCC suspected patients in the Slovak population by investigating microsatellite instability in colorectal tumor tissues. MSI was studied at 5-11 loci in matched tumor and normal DNA using radioactively labeled PCR products separated on sequencing gels. High microsatellite instability (MSI-H) was present only in patients younger than 50 years, in 100% of patients having two affected relatives by colorectal cancer and in 67% of patients with only one affected relative. In both groups of patients colorectal cancer was present in two successive generations. No MSI-H was found in the group of patients older than 50 years, even if they had positive family history for colorectal cancer. Among all markers used, the BAT26 mononucleotide repeat (100%), DI0S197 and D13S175 (62.5%) dinucleotide repeats were the most frequently altered in the tumor tissues. Retrospective analysis revealed that some of the patients having MSI-H tumors have had clinicopathological characteristics frequently reported to HNPCC. The family members of those patients with MSI-H are enrolled in preventive health care program until mutational analyses will enable to select carriers from non-carriers of mutated MMR genes.

Inducible stable DNA replication (iSDR) and the uvr-dependent tolerance of pyrimidine dimers in UV-irradiated Escherichia coli are two uncoupled processes.

Inducible stable DNA replication (iSDR) is dependent on recombination and is supposed to play a role in DNA repair of Escherichia coli. Our previous data suggested that iSDR may be involved in the tolerance of UV lesions, which remain unexcised in excision-proficient E. coli exposed to some UV pretreatments. Now, the tolerance of unexcised lesions has been followed in E. coli recB21 and in E. coli priA1 sup mutants, incapable of iSDR. The obtained data do not confirm the previous hypothesis about the involvement of iSDR in the putative uvr-dependent lesion tolerance. They rather suggest that iSDR and the uvr-dependent lesion tolerance are two uncoupled processes.

Lack of correlation between repair of DNA interstrand cross-links and differential sensitivity of G0 and proliferating CD4+ lymphocytes towards cisplatin.

G0 cells in a tumor are insensitive to the chemotherapeutical agents. The nature of this resistance is not completely understood. One of the factors modulating sensitivity of cells may be DNA repair of drug induced DNA damage. In this study we have compared gene-specific formation and repair of cisplatin-induced interstrand cross-links (ICL) in human G0 and proliferating CD4+ lymphocytes. Cisplatin killing of G0 CD4+ lymphocytes is inefficient, and these cells resemble those in a tumor. After exposure to cisplatin under similar conditions, the frequency of ICL introduced is twice as high in the proliferating compared to the resting lymphocytes. Repair of ICL was measured in the housekeeping gene, dihydrofolate reductase (DHFR), in the proliferation inducible c-myc gene, and in the inactive delta-globin gene. We observed similar relative rates and extent of ICL repair in all three genes studied, in G0 or proliferating CD4+ lymphocytes. The mechanisms responsible for the resistance of G0 CD4+ lymphocytes towards cisplatin are discussed.

The pre-UV nutritional stresses increase UV resistance, decrease UV mutagenesis and inhibit excision repair.

Nutritional stresses applied to E. coli prior to UV irradiation increase UV resistance and decrease UV mutagenesis. This effect is uvrA-dependent and might reflect a more efficient excision of pyrimidine dimers [1]. The data presented here, however, indicate that after prestarvation for glucose or amino acids pyrimidine dimer excision (PDE) was partly inhibited. It appears that the stress conditions stimulate a mode of uvr-dependent tolerance of lesions, efficient and precise. Possible modes of PDE inhibition and lesion tolerance are discussed.

The effect of the OmpT protease on excision repair in UV-irradiated Escherichia coli.

The extent of pyrimidine dimer excision (PDE) was inhibited in UV-irradiated E. coli KS272 (ompT+) cells when they were preinduced by a low UV predose preceded by a nutrition stress but not in the preinduced E.coli SF100 (ompT-) mutants. The preinduction, however, markedly inhibited PDE in the ompT- cells transformed with a multicopy plasmid carrying ompT gene. The data are consistent with the hypothesis that the inducible OmpT protease (controlled by rpoH) might terminate the SOS period of excision repair so that when cells are preinduced PDE might be inhibited prematurely.

Gene-specific repair in human CD4+ lymphocytes reflects transcription and proliferation.

We have measured the gene-specific repair of ultraviolet irradiation (UV)-induced cyclobutane pyrimidine dimers (CPD) in freshly isolated human peripheral blood CD4+ T-lymphocytes. Two populations of CD4+ lymphocytes were assayed: resting and proliferating cells. DNA repair was assessed in the essential gene dihydrofolate reductase (DHFR) as well as in each of its strands, in the proliferation inducible c-myc gene and in the inactive delta-globin gene. Transcription rates in these genes were determined by nuclear run-on assay in the two cell populations. The rate of DHFR transcription increased 10-fold from resting to proliferating lymphocytes. Transcripts from c-myc were present only in proliferating cells, and we detected no delta-globin transcripts in either cell population. During the 24-h period after UV irradiation, there was little or no repair in any of the genes in the resting cells; there was some repair in the transcribed strand of the DHFR gene, but no repair in its nontranscribed strand. In the proliferating cells where the transcription of DHFR was much increased, the repair was efficient. The delta-globin gene was not expressed in either cell population, but it was more efficiently repaired in the proliferating than in the resting cells. We suggest that the gene-specific repair activity in CD4+ lymphocytes can reflect the proliferative state of the cells as well as the transcriptional state of the gene.

Constitutive increase of RecA protein: its influence on pyrimidine dimer excision and survival of UV-irradiated Escherichia coli.

Transformation of E. coli with the plasmid pRA containing recA gene increased the constitutive level of RecA protein 50-67 fold. This slightly inhibited pyrimidine dimer excision and reduced cell survival in three investigated, UV-irradiated E. coli strains. Our data support the view that RecA protein prematurely present at a high level may mask the dimers. The masking subsequently reduces the dimer excision and switches off the inducing signal.

Inducible stable DNA replication of Escherichia coli tolerates unexcised pyrimidine dimers in an uvr-dependent manner.

Damage-inducible DNA replication (iSDR) was followed in UV-irradiated E. coli uvr+ and uvr B5 cells. Owing to the inhibition of dimer excision in the former (caused by the metabolic treatment), both contained similar amounts of unexcised dimers. Since the iSDR took place in uvr+ but not in uvr B5 cells, it is concluded that the uvr system can tolerate unexcised dimers through the recombinogenic iSDR.

DNA repair in Escherichia coli: the dual function of uvr genes.

It has been shown earlier that the starvation of E. coli for both amino-acids and thymine applied prior to UV irradiation inhibits pyrimidine dimer excision without affecting cell survival after UV irradiation. In such cells pyrimidine dimers are tolerated by a rather error-free process that depends on the activity of uvrB, recA and lexA genes. Data presented here show: (a) that the efficient toleration of unexcised dimers requires also the uvrA gene; (b) that the starvation increases the level of RecA protein about 4.7 times; (c) that the effect of starvation on subsequent pyrimidine dimer excision is reversed by a 2 h incubation in complete medium before the cells are UV irradiated. The data suggest that the uvrA, uvrB, recA, lexA dependent nonexcisional repair may be a pathway temporarily functioning in repeatedly damaged cells.

Methyl methanesulfonate adduct formation and repair in the DHFR gene and in mitochondrial DNA in hamster cells.

Chinese hamster ovary CHO-B11 cells were exposed to methyl methanesulfonate (MMS) and the formation and repair of N-methylpurines were measured in the endogenous dihydrofolate reductase (DHFR) gene domain and in mitochondrial DNA by alkaline hydrolysis which generates strand breaks at apurinic (AP) sites formed after neutral depurination. The initial levels of damage in the transcriptionally active DHFR gene, 3'-flanking non-transcribed region and in mitochondrial DNA were slightly different; the highest level of damage was in mitochondrial DNA, the lowest in the DHFR gene. The rate of adduct removal was similar in all three DNA regions examined. We conclude that there is no preferential repair of MMS induced N-methylpurines under these conditions, and that these lesions are efficiently removed from mitochondrial DNA.

Repair of ribosomal RNA genes in hamster cells after UV irradiation, or treatment with cisplatin or alkylating agents.

We have measured the DNA damage formation and repair in the ribosomal and the dihydrofolate reductase (DHFR) genes after treatment of hamster cells with different types of DNA damaging agents. In mammalian cells, the ribosomal DNA (rDNA) is transcribed by RNA polymerase I, whereas the DHFR is transcribed by RNA polymerase II, whereas the DHFR is transcribed by RNA polymerase II. Cells were treated with agents that induce different types of lesions, and that are known to be repaired via different pathways. We used UV (254 nm) irradiation, treatment with cisplatin and treatment with the alkylating agents nitrogen mustard (HN2) and methyl methanesulphonate (MMS). UV induced pyrimidine dimers were detected with the enzyme T4 endonuclease V, which creates nicks at the dimer sites; the breaks are then resolved and identified by denaturing electrophoresis and Southern blot. Intrastrand adducts formed by the alkylating agents HN2 and MMS were quantitated by generating strand breaks at abasic sites after neutral depurination. Interstrand crosslinks (ICL) formed by HN2 and cisplatin were detected by a denaturation-reannealing reaction before neutral agarose gel-electrophoresis. We find that the repair of the pyrimidine dimers is significantly less efficient in the RNA polymerase I transcribed rDNA genes than in RNA polymerase II transcribed DHFR gene at 8 and 24 h after irradiation. ICL and intrastrand adducts induced by HN2 are also removed more slowly from the rDNA than from the DHFR gene. In contrast, MMS induced intrastrand adducts and cisplatin induced ICL are repaired equally efficiently in the RNA polymerase I and RNA polymerase II transcribed genes. We conclude that for some types of DNA damage, there is less repair in the ribosomal genes than in the DHFR; but for other DNA lesions there is no difference. The difference in repair efficiency between the rDNA and the DHFR genes may reflect the different RNA polymerase involved in their transcription. It may, however, alternatively, reflect the different nuclear localization of these genes.

Overexpression of metallothionein in Chinese hamster ovary cells and its effect on nitrogen mustard-induced cytotoxicity: role of gene-specific damage and repair.

Overexpression of metallothionein in mammalian cells has been associated with protection from cytotoxic chemicals and acquired resistance of tumors to cytotoxic drugs. The mechanism of this effect, however, remains unclear. We have explored whether cytotoxicity of the bifunctional alkylating agent nitrogen mustard was correlated with the extent of DNA damage formation and repair in the metallothionein gene regions in Chinese hamster ovary cells. The DNA damage and repair were examined in metallothionein-overexpressing, cadmium-resistant Chinese hamster ovary cells, Cdr200T1, with or without zinc-induced transcriptional activation, and in the parental CHO-met- cell line. The zinc-induced Cdr200T1 cells tolerated significantly higher doses of nitrogen mustard than did the uninduced Cdr200T1 variant. The parental CHO-met- cells, which did not have any detectable metallothionein expression, were even more resistant to nitrogen mustard than the zinc-induced Cdr variants. Nitrogen mustard-induced N-alkylpurines were formed with a higher frequency in inactive genomic regions than in the active genes. The removal of N-alkylpurines was similar in the active MT I gene region in Cdr200T1 and the silent MT I gene region in the parental cells, and the expression of these genes was determined by Northern assay. The MT II gene-containing region was repaired less efficiently than the MT I gene, independently of zinc induction. Further, preferential repair of nitrogen mustard-induced N-alkylpurines were detected in a single copy of the essential active dihydrofolate reductase gene as compared to a downstream noncoding region. This preferential repair was unaffected by the presence of zinc. Neither damage formation nor repair kinetics in the MT gene regions seemed to parallel the observed spectrum of sensitivity to HN2.

Expression of the Escherichia coli recA gene in the yeast Saccharomyces cerevisiae.

The isolation of the protein coding region of the recA gene from Escherichia coli by extensive Bal31 digestion is described. The structural recA gene was ligated into an extrachromosomally replicating yeast expression vector, downstream of the yeast alcohol-dehydrogenase gene promoter region, to produce pADHrecA plasmid. The pADHrecA plasmid was transformed into the wild-type and the repair deficient strains of Saccharomyces cerevisiae. The crude protein samples were extracted from the individual yeast transformants. A 38 kDa protein was present in all transformants containing the recA gene on plasmid. Thus the recA gene from E coli was successfully expressed in cells from a lower eukaryote.

Polyomavirus-based shuttle vectors for studying mechanisms of mutagenesis in rodent cells.

We have constructed a series of polyomavirus-based shuttle vectors for analyzing mechanisms of mutagenesis in rodent cell systems. These vectors contain the supF suppressor tRNA gene which serves as the mutagenesis target; the pBR327 replication functions and ampr gene for replication and selection in bacteria; and the polyomavirus genome which permits replication in rodent cells. The polyoma genomes used in these vectors vary in their enhancer regions, causing varying efficiencies of replication in different types of rodent cells. One of the vectors (pPySLPT-2) which replicates particularly well in several different rodent cell types (i.e., Chinese hamster ovary, mouse hepatoma and mouse lymphoma) was used to compare mutation induction by UV radiation in UV repair-deficient mouse lymphoma L5178Y-R cells with mutagenesis in the related UV repair-proficient line, L5178Y-S. In both cell types, UV-induced mutants could be recovered at frequencies up to 50-fold higher than that of the spontaneous background. At a given UV fluence the L5178Y-R cells were more highly mutable than the L5178Y-S cells. Our results indicate that these new polyomavirus-based vectors should be useful for analysis of the molecular mechanisms of mutation induction in rodent cell systems, and in particular should allow detailed analysis of mutagenesis in the well characterized rodent somatic cell mutants.

Transient appearance of photolyase-induced break-sensitive sites in the DNA of ultraviolet light-irradiated Syrian hamster fetal cells.

Syrian hamster fetal fibroblasts (HFC) were examined for photolyase-induced break-sensitive sites after ultraviolet light (UV) exposure and growth. These sites, observed in excision-defective human xeroderma pigmentosum (XP) cells, are due to cleavage of the internal phosphodiester bond of UV-induced pyrimidine dimers. Excision-inefficient HFC acquired photolyase-induced break-sensitive sites during incubation after UV (10 J/m2). However, these were observed transiently, with a maximum of 5% of the pyrimidine dimers at 9 h post UV; by 18 h they were undetectable. Caffeine (1 mM) delayed the peak of photolyase-induced break-sensitive sites by 2 h. In human XP cells photolyase-induced break-sensitive sites accumulate to a plateau level of about 20% of the pyrimidine dimers. The present results extend to rodent cells the observation that cleavage of the internal phosphodiester bond of pyrimidine dimers may be an early step in their excision repair. Furthermore, the data suggest that photolyase-induced break-sensitive sites might be necessary for replication bypass at pyrimidine dimers.

Error-free uvr+-dependent inducible DNA repair in Escherichia coli B/r Hcr+ cells.

The frequency of suppressor (tryptophan reversions) and of true (streptomycin-resistant and dependent) mutations has been followed in E. coli cells irradiated with a single dose or two separate doses of ultraviolet (U.V.) radiation. Under these conditions dimers were efficiently excised after a single dose, while about 40 per cent of the dimers remained unexcised after two doses. Although the level of unexcised dimers in the latter case increased proportionally with the second U.V. dose, the mutation frequency increased by 1.5-2-fold, but did not continue to increase with the level of unexcised dimers. A comparison of excision-proficient and excision-deficient cells containing similar amounts of persisting dimers has shown that proficient cells can tolerate a high level of dimers without an adequate increase in mutation frequency. Our results suggest the existence of an error-free uvr+-dependent inducible repair in E. coli B/r Hcr+ cells.

DNA of Chinese hamster V79 cells newly synthesized after ultraviolet irradiation contains alkali-labile sites.

Pulse-labeled daughter DNA of UV-irradiated Chinese hamster V79 cells was denatured in alkaline or neutral conditions and analysed by sucrose gradient centrifugation. A comparison of the sedimentation profiles of DNA treated in alkaline or neutral conditions has shown that in UV-irradiated cells some alkali-labile sites are produced during replication of damaged templates.