A.A. van Zeeland

Molecular mechanisms involved in the production of cromosomal aberrations II. Utilization of neurospora endonuclease for the study of aberration production by X-rays in G1 and G2 stages of the cell cycle

Chinese hamster ovary cells (CHO) were X-irradiated in G1 and G2 stages of the cell cycle and subsequently Neurospora endonuclease (NE) (E.C.3.1.4), an enzyme which is specific in cleaving single-stranded DNA, was introduced into the cells, after making the cells permeable by treatment with inactivated Sendai virus. With this treatment all classes of X-ray-induced chromatid aberrations increased in G2 cells, whereas in G1 cells an increase in chromosome type of aberrations was found, associated with a profound induction of chromatid type of aberrations as well. Duration of the availability of single-strand gaps for the action of NE has been studied in G2 cells following X-irradiation and the influence of different parts of the G2 stage on the type and frequencies of chromatid aberrations was discerned. While the increase in chromosome type of aberrations by NE in X-irradiated G1 cells has been interpreted as due to the conversion of DNA single-strand breaks or gaps to double-strand breaks by NE, the induction of chromatid aberrations in G1 has been assumed to be due to conversion of some of the damaged bases into strand breaks by NE. Biochemical evidence is presented for the conversion by NE of DNA single-strand breaks induced by X-rays into double-strand breaks using neutral sucrose gradient centrifugation.

Transcription-coupled repair removes both cyclobutane pyrimidine dimers and 6-4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in xeroderma pigmentosum group C fibroblasts.

We investigated the contribution of the global and the transcription‐coupled nucleotide excision repair pathway to the removal of structurally different DNA lesions. The repair kinetics of UV‐induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6‐4) pyrimidone photoproducts (6‐4PPs) were determined in an active and inactive gene in normal human fibroblasts and in xeroderma pigmentosum group C (XP‐C) fibroblasts. Previously we have shown that in normal human cells exposed to a UV dose of 10 J/m2 repair of CPDs takes place via two pathways: global repair and transcription‐coupled repair, the latter being responsible for accelerated repair of CPDs in the transcribed strand of active genes. So far, no clear evidence for transcription‐coupled repair of 6‐4PPs has been presented. Here we demonstrate that 6‐4PPs really form a target for transcription‐coupled repair. In XP‐C cells, exposed to 30 J/m2 and only capable of performing transcription‐coupled repair, CPDs as well as 6‐4PPs are removed selectively and with similar kinetics from the transcribed strand of the adenosine deaminase (ADA) gene. The non‐transcribed strand of the ADA gene and the inactive 754 gene are hardly repaired. In contrast to XP‐C cells, normal cells exposed to 30 J/m2 lack strand‐specific repair of both 6‐4PPs and CPDs, suggesting that transcription‐coupled repair is overruled by global repair, probably due to severe inhibition of transcription at this high UV dose. The much more rapid repair of 6‐4PPs compared with CPDs in normal cells may be related to higher affinity of the global repair system for the former lesion. In XP‐C cells the similarity of the rate of repair of both 6‐4PPs and CPDs in the transcribed strand at 30 J/m2 indicates that transcription‐coupled repair of photolesions takes place in a sequential way. Our results strongly suggest that the significance of transcription‐coupled repair for removal of lesions depends on the type of lesion and on the dose employed.

Linear dose —response relationships after prolonged expression times in V-79 Chinese hamster cells

The expression time for induced mutants resistant to 6-thioguanine, in V-79 Chinese hamster cells, was determined by respreading the cells in the selective medium, at various times after treatment. The length of the expression time for mutants induced by X-rays, ethyl methane sulphonate and ultraviolet irradiation was dose dependent. For the highest dose used this was 7 to 8 days, beyond which there was no further changes in mutant frequency. The dose-response relationship of these agents does not appear to deviate from linearity; this permits the calculation of mutation rate per unit dose. For X-rays this value was 1.35 - 10(-7) per rad per locus, for ethyl methane sulphonate, 2.2 - 10(-2) per mole per locus and for ultraviolet irradiation, 6.3 - 10(-6) per erg per mm2 per locus. The effectiveness of the 3 different mutagens for the induction of mutations was compared by calculating the increase in mutant frequency per unit of decrease in survival (Do). These increments in frequency were: 5.6 - 10(-5) for X-rays, 69.5 - 10(-5) for ethyl methane sulphonate and 16.1 - 10(-5) for ultraviolet irradiation.

DNA strand specificity for UV-induced mutations in mammalian cells

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

Contribution of incorporated 5-bromodeoxyuridine in DNA to the frequencies of sister-chromatid exchanges induced by inhibitors of poly-(ADP-ribose)-polymerase

3-Aminobenzamide and benzamide, two potent inhibitors of poly-(ADP-ribose)-polymerase increase the frequencies of SCEs in Chinese hamster ovary cells in a dose-dependent manner. SCEs were studied in cells in which the inhibitors were present either during the first cell cycle or the second cell cycle or both. Most of the induced SCEs were found to be formed during the second cell cycle in which BU-containing DNA was used as template for DNA synthesis. In cells which were pregrown for 4 cell cycles in the presence of BrdUrd, in order to obtain both sister chromatids bifiliarly substituted with BU in their DNA, it was found that the presence of inhibitor even in the first cell cycle increased the frequencies of SCEs. It is concluded that the incorporated BrdUrd plays an important role in the origin of spontaneous and induced SCEs. 3-Aminobenzamide alone or benzamide in the presence of BrdUrd during culture, did not increase the frequencies of mutations to HGPRT- in these cells.

The sensitivity of Cockayne's syndrome cells to DNA-damaging agents is not due to defective transcription-coupled repair of active genes.

Two of the hallmarks of Cockaynes syndrome (CS) are the hypersensitivity of cells to UV light and the lack of recovery of the ability to synthesize RNA following exposure of cells to UV light, in spite of the normal repair capacity at the overall genome level. The prolonged repressed RNA synthesis has been attributed to a defect in transcription-coupled repair, resulting in slow removal of DNA lesions from the transcribed strand of active genes. This model predicts that the sensitivity of CS cells to another DNA-damaging agent, i.e., the UV-mimetic agent N-acetoxy-2-acetylaminofluorene (NA-AAF), should also be associated with a lack of resumption of RNA synthesis and defective transcription-coupled repair of NA-AAF-induced DNA adducts. We tested this by measuring the rate of excision of DNA adducts in the adenosine deaminase gene of primary normal human fibroblasts and two CS (complementation group A and B) fibroblast strains. High-performance liquid chromatography analysis of DNA adducts revealed that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the main adduct induced by NA-AAF in both normal and CS cells. No differences were found between normal and CS cells with respect to induction of this lesion either at the level of the genome overall or at the gene level. Moreover, repair of dG-C8-AF in the active adenosine deaminase gene occurred at similar rates and without strand specificity in normal and CS cells, indicating that transcription-coupled repair does not contribute significantly to repair of dG-C8-AF in active genes. Yet CS cells are threefold more sensitive to NA-AAF than are normal cells and are unable to recover the ability to synthesize RNA. Our data rule out defective transcription-coupled repair as the cause of the increased sensitivity of CS cells to DNA-damaging agents and suggest that the cellular sensitivity and the prolonged repressed RNA synthesis are primarily due to a transcription defect. We hypothesize that upon treatment of cells with either UV or NA-AAF, the basal transcription factor TFIIH becomes involved in nucleotide excision repair and that the CS gene products are involved in the conversion of TFIIH back to the transcription function. In this view, the CS proteins act as repair-transcription uncoupling factors. If the uncoupling process is defective, RNA synthesis will stay repressed, causing cellular sensitivity. Since transcription is essential for transcription-coupled repair, the CS defect will affect those lesions whose repair is predominantly transcription coupled, i.e., UV-induced cyclobutane pyrimidine dimers.

Relationship between cell killing, chromosomal aberrations, sister-chromatid exchanges and point mutations induced by monofunctional alkylating agents in Chinese hamster cells a correlation with different ethylation products in DNA

Several monofunctional alkylating agents (AA) were compared for their ability to induce chromosomal aberrations, cell killing, sister-chromatid exchanges (SCE) and point mutations in Chinese hamster cells (CHO and V79 cells). The AAs chosen varied in their reaction kinetics as well as their affinity to nucleophilic sites (different s values). AAs with low s values were more mutagenic in comparison to those with high s values, whereas the reverse was true for induction of cytotoxic effects. Neither SCEs nor chromosomal aberrations correlated with the induction of point mutations, indicating that different primary DNA lesions and repair pathways are involved in these biological processes. Molecular dosimetric studies indicate that O6 alkylation of guanine is the most probable cause of lesions in DNA leading to point mutations following treatment with ethyl methanesulphonate and ethyl nitrosourea.

Sensitive determination of pyrimidine dimers in DNA of UV-irradiated mammalian cells Introduction of T4 endonuclease V into frozen and thawed cells

Endonuclease V from E. coli infected with phage T4 was used to evaluate the frequency and the removal of pyrimidine dimers from DNA in cultured mammalian cells. Cellular membranes were made permeable to the enzyme by two cycles of rapid freezing and thawing. The number of endonuclease-sensitive sites in DNA was assayed by sedimentation in alkaline sucrose gradients upon which the cells were lysed directly. Comparison of the frequency of endonuclease-sensitive sites with the frequency of pyrimidine dimers determined by chromatographic analysis of hydrolysed DNA indicated that about 50% of the dimers in the permeabilized cells were substrates for T4 endonuclease V. This was confirmed by observation that when DNA treated with the enzyme in situ was purified, it contained the expected additional number of endonuclease-sensitive sites if again treated with the enzyme. The percentage of pyrimidine dimers recognized by T4 endonuclease V was enhanced to nearly 100% by exposing the permeabilized cells to 2 M NaCl before the enzyme was introduced. This method allowed the measurement of frequencies of endonuclease-sensitive sites after doses of UV irradiation at low as 0.5 J/m2. Loss of endonuclease sites from cellular DNA was observed during post-irradiation incubation of V79 Chinese hamster cells and several human cell strains. A comparison of the results obtained in human cells with or without the high-salt exposure before endonuclease treatment suggested that the dimers recognized under low-salt conditions may be removed slightly faster than those recognized only after high-salt exposure.

Mutations induced by X-rays at the HPRT locus in cultured Chinese hamster cells are mostly large deletions.

We investigated the molecular basis of 19 X-ray-induced HPRT-deficient mutants of V79 Chinese hamster cells with Southern hybridisation techniques. 12 of those mutants suffer from a big deletion (greater than 10 kb) of HPRT DNA sequences. Cytological studies of chromosome preparations of those 12 deletion mutants showed that in at least 3 of these mutants part of the long arm of the X-chromosome was lost. After correction for spontaneous arising mutations we estimate that at least 70-80% of X-ray-induced mutations are caused by large deletions.

The role of metabolic cooperation in selection of hypoxanthine-guanine-phosphoribosyl-transferase (HG-PRT)-deficient mutants from diploid mammalian cell strains

Abstract A system for the selection of 8-azaguanine-resistant mutants from a diploid human cell strain is described. The selection of mutant cells is largely influenced by a phenomenon, known as metabolic cooperation, which turns mutant cells into phenotypically wild-type cells. As a consequence mutant cells cannot be selected above a certain cell density. Reconstruction experiments in which mutant cells were mixed with wild-type cells and mutant feeder cells showed that a reasonable recovery of mutant cells could only be obtained at a density of about 190 wild-type cells per cm 2 . Under these conditions HG-PRT (hypoxanthine-guanine-phosphoribosyl-transferase) deficient clones were obtained from a diploid human skin fibroblast strain and from a mouse skin fibroblast strain. In order to improve the selection system the mechanism of metabolic cooperation should be understood. Therefore it was investigated whether metabolic cooperation is due to cell-to-cell contact or to factors mediated by the medium. It is shown that when mutant cells and wild-type cells were separated by a fibrin layer, metabolic cooperation did not occur.