Description of research work:
The project aims at understanding the basis of radiation sensitivity in humans, and the underlying role of DNA repair. Such knowledge is important for assessing relative radiation risk from one individual to another because people do not all respond equally to radiation.
Research has been carried out in 3 areas:
Deoxyribonucleic acid (DNA) repair and chromatin structure:
Cyclobutane pyrimidine dimers (CPD) are preferentially removed from transcriptionally active DNA in ultraviolet (UV) irradiated hamster and human cells. In V79 hamster cells repair of CPD in the active hypoxanthine phosphoribosyltransferase (HPRT) gene is mainly confined to the transcribed strand, whilst, in human cells repair of CPD in housekeeping genes is faster in the transcribed strand. In human cells CPD repair of X-chromosomal repressed genes is less efficient than in housekeeping genes, suggesting heirarchies of DNA repair in mammalian cells. Different levels of CPD repair in transcriptionally active and inactive DNA sequences are found in cells derived from UV sensitive genetic disorders (Xeroderma pigmentosum, Cockayne's Syndrome).
Encapsulated cells have been used to isolate intact chromatin as a template for in vitro repair synthesis. In exponentially growing human cells, in vitro replication is inhibited by UV radiation. In normal human fibroblasts, incorporation by replication is sufficiently low to detect UV induced repair synthesis.
Mutation spectra in repair proficient and deficient cells:
All types of transitions are present in repair proficient cells whereas spectra of repair deficient cells are dominated by guanine cytosine-adenine thymine (GC-AT) transitions. In UV irradiated repair proficient cells mutations are generated predominantly by damage sites in the poorly repaired nontranscribed strand whereas, in repair deficient cells, mutations are generally in the transcribed strand.
Characterization of UV sensitive mutants:
UV sensitive mutants of hamster cells have been studied and results suggest that 6-4 PP is the main mutagenic lesion.
New techniques have been developed for measuring cellular radiosensitivity. T-lymphocytes have been found to provide the most rapid (approximately 14 days from blood separation) estimate of radiosensitivity based on a clonal assay. T-lymphocytes from neonatal cord blood have been found to be significantly more radiosensitive than T-lymphocytes from adult blood. T-lymphocytes from ataxia-telangiectasia (A-T) donors are also more radiosensitive. Work has been carried out on a single cell microgel assay for fast results (within 1 day). Attempts have also been made to couple the repair of potentially lethal damage (RPLD) experimental design to the study of the repair of clastogenic damage by measuring changes in the induction of micronucleic in fibroblasts. A-T heterozygotes are defective in the repair of this damage. A-T heterozygotes are cancer prone and results from premenopausal breast cancer patients show a substantial number are defective in repair of clastogenic damage.
Experiments have also been carried out on the cloning of dexoyribonucleic acid (DNA) repair genes. A search in A-T cell strains for deletions of DNA containing known markers using pulse field gel electrophoresis (PFGE) detected no deletions. Hamster xrs mutants are sensitive to ionizing radiation. An attempt has been made to localise the human xrs gene using microcell-mediated chromosome transfer. Human chromosome 2 restores radiation resistance suggesting the xrs gene is associated with chromosome 2. The fission yeast Schizosaccharomyces pombe has also been employed in cloning DNA repair genes and from comparisons with S cerevisiae, functional domains have been identified. The cloned genes have been mapped on the S pombe chromsomes using PFGE.
Research has been carried out into the isolation and characterization of human genes involved in excision repair. Using deoxyribonucleic acid (DNA) mediated gene transfer to the repair deficient rodent mutant 27-1, the human excision repair gene ERCC-3 has been isolated. This gene corrects the ultraviolet (UV) sensitivity and unscheduled DNA synthesis (UDS) of mutants belonging to complementation group 3. The gene (chromosomal location 2q21) is 45 kb and consists of 15 exons and specifies a protein of 782 amino acids. A correcting human gene, ERCC-6, has been cloned using a Chinese hamster mutant cell line, UV61, which is UV sensitive and nucleotide excision repair deficient. The ERCC-6 gene (chromosomal location 10q11-21.1) is more than 80 kb and encodes a protein of at least 1500 amino acids. It may be implicated in Cockayne's Syndrome (CS). The Saccharomyces cerevisiae RAD6 gene is implicated in postreplication repair, damage induced mutagenesis and sporulation. 2 human RAD6 homologs, HHR6A and HHR6B, have been cloned and carry out similar functions to RAD6. The HHR6A gene was localised on the X chromosome (q26-25) and the HHR6B gene was assigned to chromosome 5q23-31.
The characterization of human repair genes and proteins is in full swing.
Research has been carried out using Fanconi anemia (FA) cells in culture for molecular analysis. The cells were found to be hypomutable at the hypoxanthine phosphoribosyltransprase (HPRT) and the sodium potassium adenosintriphosphatase (ATPase) loci compared to normal. This was true for the 2 genetic complementation groups A and B. Analysis of spontaneous and induced mutants showed that, in FA cells, deletion mutants predominate whereas, in normal cells, the majority of mutants were due to point mutations. The hypomutability in FA cells associated with a reduction in frequency of point mutations suggests FA cells are defective in a mutagenic process operating in normal cells. Studies have been carried out on the correction of the FA defect by cocultivation with normal human or mouse cells and cloning of complementary dexoyribonucleic acid (cDNA) has been performed. Anomalies have also been found in the regulation of production of at least 2 cytokines, interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-alpha), and further work will investigate the relation between these and the DNA repair defect.
The efficiency and fidelity of rejoining of site specific dexoyribonucleic acid (DNA) double strand breaks in a cell free system has been determined using extracts from normal and ataxia-telangiectasia (A-T) cell lines. The efficiency of rejoining did not vary with the cell type used for extract preparation but the fidelity of rejoining was poor at some sites and with some extracts. In particular, at sites with a low rejoin efficiency, the fidelity of rejoining was much lower for the A-T extracts than for normal cell extracts. At other sites, misrejoining was unrelated to rejoin efficiency, suggesting that factors such as the exact sequence at the break site may influence fidelity of rejoining.
The ability of the radiosensitive mutant irs2, which has a similar phenotype to A-T cells, to recover from damage under low dose rate irradiation has been assessed. Like A-T cells, the irs2 mutant showed little recovery under protracted irradiation. Further studies aim to map the human gene complementing irs2.
A method has been developed for analysis of the molecular nature of large deletions in the hypoacanthine phosphoribosyltransferase (HPRT) gene in early passage male human cells and breakpoints are being assessed using the polymerase chain reaction (PCR).
Ionising radiation (IR) complementation group 5 mutants, represented by xrs1-6 and XR-V15B, from the hamster CHO and V79 cell lines respectively, are highly sensitive to IR and defective in rejoining double strand breaks (DSB). IR group 5 mutants are hypermutable compared to parental cells following exposure to the same radiation dose. They are also hypersensitive to the induction of chromosome aberrations by IR. This suggests that the unrejoined breaks in group 5 mutants can be aberrantly rejoined to yield mutations and exchanges between chromosomes. In contrast, some forms of recombination occur at decreased frequencies in group 5 mutants, including V(D)J recombination. A mechanism is proposed whereby the xrs gene product acts as part of a complex to protect free ends and/or to promote their ligation. Thus the ends of DSB must be held together by a complex to promote accurate ligation. If such a complex does not form, the ends may be subjected to nuclease digestion prior to ligation or ligated to other broken ends to yield exchanges between chromosomes. V(D)J recombination may also recruit this complex to protect the ends formed at the site specific DSB. While xrs cells have a normal ability to ligate both single strand (SS) and DS termini, this does not eliminate a role for the xrs gene product in enhancing the ligation process or protecting free ends. It is possible that the enhanced mutations and rearrangement seen in xrs cells following irradiation could cause the changes necessary for carcinogenesis or even that aberrant V(D)J recombination events might result in such an outcome.
In vitro systems have been used to elucidate the mechanisms of deoxyribonucleic acid (DNA) double strand break (DSB) repair. DNA molecules with a site specific break were incubated with cellular extracts and the products analysed by bacterial transformation studies. It was found that a DNA end with a 2 basepair (bp) overhang could be greatly stimulated by the presence of polyethylene glycol (PEG), and also that increasing the ionic strength of the reaction could greatly increase the amount of circular products produced. These 2 agents may be useful in the production of either circles or linear multimers depending on the products of interest, but the production of circular products was suppressed when the 2 were used in combination. It was also found that a proportion of molecules are misrejoined by extract treatment, through a mechanism involving short sequence repeats. Attempts to reconstruct this misrejoin process suggest that multiple cellular components are involved.
A link has been proposed between damage produced by reactive oxygen species, ageing and cancer. It has been further suggested that some 'life style' effects on the incidence of cancer might relate to dietary antioxidant levels giving protection against oxidative damage to deoxyribonucleic acid (DNA). Since ionising radiation damage is also mediated by radical attack, is sensitive to cellular oxygen levels and is at least partly mediated by active oxygen species it might also be influenced by dietary levels of antioxidants. Blood samples were taken after donors had fasted overnight and again one hour after they had eaten breakfast and taken approximately 35 mg/kg vitamin C. We measured initial DNA damage using the Comet assay (single cell gel electrophoresis), in which DNA single strand breaks (SSB) generate a comet tail streaming from the nucleus. In repeat experiments on 6 donors a reduction in damage, as indicated by a highly significant decrease in overall comet length, was observed following vitamin C ingestion, both in the unirradiated controls and in the dose response to ionising radiation. Consistent differences between donors were also found. The peak effect was 4 hours after intake of food and vitamin C. The effect persisted for more than 6 hours but not overnight. An effect was also seen after breakfast without additional vitamin C. These results refer only to the Comet assay and more extensive work is required, with other end points such as cell survival and clastogenicity.
The mutagenic potential of double strand breaks (DSB) generated by restriction endonucleases (RE) in mammalian cells has been investigated. The mutation target was a single copy of the bacterial gpt gene stably integrated into the genome of Chinese hamster ovary cells. A large number of RE were screened covering a range of cutting frequencies and types of DSB termini (eg blunt, or staggered ends with 3' or 5' overhang of varying length). No clear correlation was found for either number of cut sites within the gene, or DSB end structure, and mutagenicity of the RE.
In the study of the RE it was found that the mutant frequencies were barely above the spontaneous level if the cell survival levels were either very high or very low. An in vitro assay revealed that in a majority of cases minimal cell kill corresponded with RE that barely showed any activity under simulated cellular conditions, while excessive cell kill corresponded with enzymes that were very active for over 24 h. Thus the extent and duration of activity of the RE, as measured under simulated cellular conditions, was a good indication of the amount of deoxyribonucleic acid (DNA) damage induced in vivo (as reflected by cell survival). Understandably, an enzyme that is hardly active in the cellular environment would be generating few DSB, if any, and therefore would not be mutagenic. Conversely, RE that are very active and long lived, thereby inducing many DSB over a long period of time, would be extremely detrimental to the cell. In this case, mutants are lost due to the lethal effects of excessive damage sustained by the rest of the genome. We found that the most mutagenic RE, Sau3A1, AluI and MspI, were enzymes that showed very high activity for short periods.
The efficacy of RE in vivo, thus appears to be determined by the extent of their activity in the cellular environment and the lifetime of the enzymes within the cell, these factors masking other parameters such as cutting frequency or type of DSB. The mutagenicity of RE would seem to be a fine balance between an ability to induce sufficient DNA damage to give a measurable effect (eg increase in mutation frequency) and protracted activity which kills the cells.
Hypoxanthine phosphoribosyltransferase (HPRT) gene mutants were isolated from primary human fibroblasts irradiated with either 250 kV X-rays or plutonium-238 alpha particles (or from unirradiated cells). Primers for the polymerase chain reaction (PCR) were created across the entire HPRT gene region (approximately 56 kb) and the presence or absence of PCR products was used to map the mutations. Many mutants were found to have large alterations, especially deletions of different parts of the gene. The breakpoints of large deletions in 5 mutants were isolated and sequenced; the most common sequence features found at the breakpoints were short sequence repeats. Many of the radiation induced deletions involved the complete loss of the HPRT gene. Some of the mutants with deletion of HPRT gene sequence were found to have codeletion of flanking probe sites in the same genomic region (Xq26), supporting earlier findings that the Xq26 regions will tolerate relatively large genetic changes. It was found that, compared to spontaneous mutants, many of the X-ray induced deletion breakpoints fell outside the gene. This may indicate that the mechanism(s) involved in the formation of these deletions are at least in part specific to X-ray induced damage. For example, these mechanisms may favour certain sequences or secondary structures in the genomic region under study. Studying the extent of deletions and the sequences at these deletion breakpoints should give information on the mechanisms involved.
Much of this proposal is concerned with hereditary differences in radiosensitivity due to homozygous effects of DNA repair and related genes, but heterozygous effects are now recognized and, because of their high frequency in the population may be more important in practical terms. The development of more sensitive and advanced techniques for determining radiosensitivity within the normal range is important and will be pursued. The collaborating laboratories will further develop the isolation and characterization of radiosensitive and DNA repair defective mutants and will continue the cloning of relevant genes following the successful cloning of the first human DNA repair gene during the last contract.
We shall also investigate what aspects of DNA sequence or chromosomal organization are important in determining reparability of DNA damage.
Funding SchemeCSC - Cost-sharing contracts
W1N 4AL London
2300 RA Leiden