Genomic integrity and the repair of double-strand DNA breaks.

The induction of double-strand breaks (DSBs) in DNA by exposure to DNA damaging agents or as intermediates in normal cellular processes, creates a severe threat for the integrity of the genome. Unrepaired or incorrectly repaired DSBs lead to broken chromosomes and/or gross chromosomal rearrangements which are frequently associated with tumor formation in mammals. To maintain the integrity of the genome and to prevent the formation of chromosomal aberrations, several pathways exist in eukaryotes: homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). These mechanisms are conserved in evolution, but the relative contribution depends on the organism, cell type and stage of the cell cycle. In yeast, DSBs are primarily repaired via HR while in higher eukaryotes, both HR and NHEJ are important. In mammals, defects in both HR or NHEJ lead to a predisposition to cancer and at the cellular level, the frequency of chromosomal aberrations is increased. This review summarizes our current knowledge about DSB-repair with emphasis on recent progress in understanding the precise biochemical activities of individual proteins involved.

Isolation and characterization of the RAD59 homologue of Kluyveromyces lactis.

Homologous recombination in the yeast Saccharomyces cerevisiae is under the control of the RAD52 epistasis group. Genes belonging to this group show strong conservation during evolution and homologues of most members have been identified in other eukaryotic organisms such as Schizosaccharomyces pombe, Drosophila and mammals. A homologue of the ScRAD59 gene, which shows structural and functional overlap with ScRAD52, has not been identified in other organisms until now. Previous assessment of the ScRAD59 function revealed that the product of this gene is required for certain types of ScRAD51-independent recombination and single-strand annealing. Also, in the distantly related fission yeast, Sch. pombe, a second RAD52 homologue has been identified (rad/22B+), but this gene more closely resembles ScRAD52 than ScRAD59 at the amino-acid level. In this study, the isolation of a homologue of ScRAD59 in Kluyveromyces lactis, KlRAD59, is described. A Klrad159 null allele results in moderate sensitivity to X-rays, indicating that the KlRAD59 gene is involved in the repair of X-ray-induced DNA damage. The amino acids in the putative K1Rad59 protein share 53% identity and 11% similarity with ScRad59. The KlRAD59 gene fully complements both the X-ray-sensitive phenotype and defects in recombination of the Scrad59 mutant strain. Our results underscore the evolutionary conservation of the RAD52 group of genes and provide evidence that the presence of additional RAD52 homologues is not limited to Sac. cerevisiae and Sch. pombe and might be a general phenomenon.

Comparison of tactile awareness in children during the production of isolated syllables of English.

To assess whether lingual-tactile feedback is developmental 60 normally developing children formed two groups of 30. Group 1 were in Grades 1 and 2 (M age=6.6 yr.) and Group 2 in Grades 5 and 6 (M age=10.7 yr.). All children passed a speech, language, and hearing screening. They were asked to imitate the production of a syllable, then describe the location of the tongue during that production. Following this request, subjects were given four multiple-choice questions to answer regarding (a) tongue height (high to low), (b) tongue position (front to back), (c) contact with the teeth, and (d) contact with other structures within the oral cavity. Seven English phonemes (t, k, sh, r, l, and th) were presented in a consonant vowel (CF) syllable with the central carat vowel (pronounced "uh"). Children were aided by a two-dimensional line drawing of the oral cavity. Mean scores for each syllable ranged from .8 to 2.0 (on a scale of 4.0). Total mean scores of 28 possible for older children (M= 11.0) was significantly better (t58=-2.2, p<.05) than that for the younger group (M=9.6). A significant r58 of .30 (p< .05) was found between age and total syllables correct. The children described tongue location during the production of isolated syllables. Older children performed the task better, indicating that lingual-tactile awareness is maturational. These findings parallel the 1967 results of McDonald and Aungst who found that identification of oral forms improved across age through about 15 years.

Complementation of chromosomal aberrations in AT/NBS hybrids: inadequacy of RDS as an endpoint in complementation studies with immortal NBS cells.

Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) are rare autosomal recessive hereditary disorders characterized by radiosensitivity, chromosomal instability, immunodeficiency and proneness to cancer. Although the clinical features of both syndromes are quite distinct, the cellular characteristics are very similar. Cells from both NBS and AT patients are hypersensitive to ionizing radiation (IR), show elevated levels of chromosomal aberrations and display radioresistant DNA synthesis (RDS). The proteins defective in NBS and AT, NBS1 and ATM, respectively, are involved in the same pathway, but their exact relationship is not yet fully understood. Stumm et al. (Am. J. Hum. Genet. 60 (1997) 1246) have reported that hybrids of AT and NBS lymphoblasts were not complemented for chromosomal aberrations. In contrast, we found that X-ray-induced cell killing as well as chromosomal aberrations were complemented in proliferating NBS-1LBI/AT5BIVA hybrids, comparable to that in NBS-1LBI cells after transfer of a single human chromosome 8 providing the NBS1 gene. RDS observed in AT5BIVA cells was reduced in these hybrids to the level of that seen in immortal NBS-1LBI cells. However, the level of DNA synthesis, following ionizing radiation, in SV40 transformed wild-type cell lines was the same as in NBS-1LBI cells. Only primary wild-type cells showed stronger inhibition of DNA synthesis. In summary, these results clearly indicate that RDS cannot be used as an endpoint in functional complementation studies with immortal NBS-1LBI cells, whereas the cytogenetic assay is suitable for complementation studies with immortal AT and NBS cells.

Isolation and genetic characterisation of the Drosophila homologue of (SCE)REV3, encoding the catalytic subunit of DNA polymerase zeta.

In Drosophila, about 30 mutants are known that show hypersensitivity to the methylating agent methyl methane sulfonate (MMS). Addition of this agent to the medium results in an increased larval mortality of the mutants. Using a P-insertion mutagenesis screen, three MMS-sensitive mutants on chromosome II were isolated. One of these is allelic to the known EMS-induced mus205 (mutagen sensitive) mutant. In the newly isolated mutant, a P-element is detected in region 43E by in situ hybridisation. The localisation of mus205 to this region was confirmed by deficiency mapping. The gene was cloned and shows strong homology to the Saccharomyces cerevisiae REV3 gene. The REV3 gene encodes the catalytic subunit of DNA polymerase zeta, involved in translesion synthesis. The P-element is inserted in the first exon of the mus205 gene resulting in an aberrant mRNA, encoding a putative truncated protein containing only the first 13 of the 2130 aa native Drosophila protein. The mus205 mutant is hypersensitive to alkylating agents and UV, but not to ionising radiation. In contrast to reported data, in germ cells, the mutant has no effect on mutability by X-rays, NQO and alkylating agents. In somatic cells, the mutant shows no effect on MMS-induced mutations and recombinations. This phenotype of the Drosophila mus205 mutant is strikingly different from the phenotype of the yeast rev3 mutant, which is hypomutable after UV, X-rays, NQO and alkylating agents.

Intrinsic genetic instability of normal human lymphocytes and its implication for loss of heterozygosity.

A combination of flow cytometry and microsatellite analysis was used to investigate loss of expression of HLA-A and/or HLA-B alleles and concurrent LOH at polymorphic chromosome 6 loci both in freshly isolated lymphocytes (in vivo mutations) and in lymphocytes cultured ex vivo. The fraction of in vivo mutants that showed LOH at 6p appeared to vary from 0%-49% for various donors. During culturing ex vivo, HLA-A(-) cells arose at a high rate and showed simultaneous loss of expression at the linked HLA-B locus. Up to 90% of the ex vivo arisen HLA-A2(-) cell population showed LOH of multiple 6p markers, and 50% had lost heterozygosity at 6q. This ex vivo spectrum resembles that found in HLA-A2 mutants obtained from lymphoblastoid cells. The HLA-A2 mutants present in vivo may reflect only a small fraction of the mutants that can be detected ex vivo. In normal lymphocytes, in vivo only mitotic recombination appears to be sustained, indicating the importance of this mechanism for tumor initiation in normal cells. Although mutations resulting in LOH at both chromosome 6 arms were shown to result in nonviable cells in normal lymphocytes, they have been shown to result in viable mutants in lymphoblastoid cells. We hypothesize that these types of mutations also occur in vivo but only survive in cells that already harbor a mutated genetic background. In light of the high rate at which these types of mutations occur, they may contribute to cancer progression.

Characterization of RAD52 homologs in the fission yeast Schizosaccharomyces pombe.

The RAD52 gene of Saccharomyces cerevisiae is essential for repair of DNA double-strand breaks (DSBs) by homologous recombination. Inactivation of this gene confers hypersensitivity to DSB-inducing agents and defects in most forms of recombination. The rad22+ gene in Schizosaccharomyces pombe (here referred to as rad22A+) has been characterized as a homolog of RAD52 in fission yeast. Here, we report the identification of a second RAD52 homolog in Schizosaccharomyces pombe, called rad22B+. The amino acid sequences of Rad22A and Rad22B show significant conservation (38% identity). Deletion mutants of respectively, rad22A and rad22B, show different phenotypes with respect to sensitivity to X-rays and the ability to perform homologous recombination as measured by the integration of plasmid DNA. Inactivation of rad22A+ leads to a severe sensitivity to X-rays and a strong decrease in recombination (13-fold), while the rad22B mutation does not result in a decrease in homologous recombination or a change in radiation sensitivity. In a rad22A-rad22B double mutant the radiation sensitivity is further enhanced in comparison with the rad22A single mutant. Overexpression of the rad22B+ gene results in partial suppression of the DNA repair defects of the rad22A mutant strain. Meiotic recombination and spore viability are only slightly affected in either single mutant, but outgrowth of viable spores is almost 31-fold reduced in the rad22A-rad22B double mutant. The results obtained imply a crucial role for rad22A+ in repair and recombination in vegetative cells just like RAD52 in S. cerevisiae. The rad22B+ gene presumably has an auxiliary role in the repair of DSBs. The drastic reduced spore viability in the double mutant suggests that meiosis in S. pombe is dependent on the presence of either rad22A+ or rad22B+.

Tactile awareness in children and adults during the production of isolated syllables of English.

The purpose of this investigation was to determine whether tactile feedback is developmental in nature. 90 subjects were placed into three groups of 30 individuals each (M ages: 6.8, 10.7, and 18.8 yr.). All subjects passed a speech, language, and hearing screening. The procedure involved asking subjects to imitate the production of a syllable, then describe where the tongue was located during that production. Following this request, subjects answered multiple-choice questions regarding (a) tongue height (high to low), (b) tongue position (front to back), (c) contact with teeth, and (d) contact with other structures within the oral cavity. Seven English phonemes (t, k, sh, r, l, and voiceless th) were presented in a consonant vowel (CV) syllable with the neutral schwa vowel. Subjects were aided by a line drawing of the oral cavity. A significant correlation was found between age and total test score. Significant differences were also found among groups on all phonemes with the exception of r and s. All subjects could describe tongue location during the production of isolated syllables. Adults performed the task best, and young children had the most difficulty, indicating that tactile awareness may be maturational. These findings parallel those of McDonald and Aungst who in 1967 found that identification of oral forms improved with age through midadolescence.

A new X-ray sensitive CHO cell mutant of ionizing radiation group 7,XR-C2, that is defective in DSB repair but has only a mild defect in V(D)J recombination.

The DNA-dependent protein kinase (DNA-PK) complex plays a key role in DNA double-strand break (DSB) repair and V(D)J recombination. Using a genetic approach we have isolated cell mutants sensitive to ionizing radiation (IR) in the hope of elucidating the mechanism and components required for these pathways. We describe here, an X-ray-sensitive and DSB repair defective Chinese hamster ovary (CHO) cell line, XR-C2, which was assigned to the X-Ray Cross Complementation (XRCC) group 7. This group of mutants is defective in the XRCC7/SCID/Prkdc gene, which encodes the catalytic subunit of DNA-PK (DNA-PKcs). Despite the fact that XR-C2 cells expressed normal levels of DNA-PKcs protein, no DNA-PK catalytic activity could be observed in XR-C2, confirming the genetic analyses that these cells harbor a dysfunctional gene for DNA-PKcs. In contrast to other IR group 7 mutants, which contain undetectable or low levels of DNA-PKcs protein and which show a severe defect in V(D)J recombination, XR-C2 cells manifested only a mild defect in both coding and signal junction formation. The unique phenotype of the XR-C2 mutant suggests that a normal level of kinase activity is critical for radiation resistance but not for V(D)J recombination, whereas the overall structure of the DNA-PKcs protein appears to be of great importance for this process.

Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine.

Somatic mutation accumulation has been implicated as a major cause of cancer and aging. By using a transgenic mouse model with a chromosomally integrated lacZ reporter gene, mutational spectra were characterized at young and old age in two organs greatly differing in proliferative activity, i.e., the heart and small intestine. At young age the spectra were nearly identical, mainly consisting of G. C to A.T transitions and 1-bp deletions. At old age, however, distinct patterns of mutations had developed. In small intestine, only point mutations were found to accumulate, including G.C to T.A, G.C to C.G, and A.T to C.G transversions and G.C to A.T transitions. In contrast, in heart about half of the accumulated mutations appeared to be large genome rearrangements, involving up to 34 centimorgans of chromosomal DNA. Virtually all other mutations accumulating in the heart appeared to be G.C to A.T transitions at CpG sites. These results suggest that distinct mechanisms lead to organ-specific genome deterioration and dysfunction at old age.

Training in lingual awareness during production of isolated syllables of English.

The purpose of this preliminary investigation was to determine the effects of training on lingual awareness during the production of isolated syllables of English. 60 subjects were selected for this investigation. They were placed into two groups of 30 individuals each. Group one (Trained) consisted of individuals who had received training in articulation and phonetics (M age=22.7) Group two (Untrained) consisted of individuals who had not received training in articulation and phonetics (M age= 18.8). The subjects in the Trained group were majors in speech-language pathology. The procedure involved asking subjects to imitate the production of a syllable, then describe where the tongue was located during that production. Following this request, subjects were given four multiple-choice questions to answer regarding (a) tongue height (high to low), (b) tongue position (front to back), (c) contact with the teeth, and (d) contact with other structures within the oral cavity. Mean scores ranged from 1.87 to 3.2 (on a scale of 4.0) for the group of Trained subjects versus 1.5 to 2.4 for the Untrained subjects. The Trained subjects had significantly higher test scores on all phonemes (p<.05) with the exception of r and sh (p>.05). For both groups of subjects, the phonemes, r, sh, and l, were the most difficult to describe, while the phoneme t was described most accurately.

Qualitative and quantitative procedures for health risk assessment.

Numerous reactive mutagenic electrophiles are present in the environment or are formed in the human body through metabolizing processes. Those electrophiles can directly react with DNA and are considered to be ultimate carcinogens. In the past decades more than 200 in vitro and in vivo genotoxic tests have been described to identify, monitor and characterize the exposure of humans to such agents. When the responses of such genotoxic tests are quantified by a weight-of-evidence analysis, it is found that the intrinsic potency of electrophiles being mutagens does not differ much for the majority of the agents studied. Considering the fact that under normal environmental circumstances human are exposed to low concentration of about a million electrophiles, the relation between exposure to such agents and adverse health effects (e.g., cancer) will become a 'Pandora's box'. For quantitative risk assessment it will be necessary not only to detect whether the agent is genotoxic, but also understand the mechanism of interaction of the agent with the DNA in target cells needs to be taken into account. Examples are given for a limited group of important environmental and carcinogenic agents for which such an approach is feasible. The groups identified are agents that form cross-links with DNA or are mono-alkylating agents that react with base-moieties in the DNA strands. Quantitative hazard ranking of the mutagenic potency of these groups of chemical can be performed and there is ample evidence that such a ranking corresponds with the individual carcinogenic potency of those agents in rodents. Still, in practice, with the exception of certain occupational or accidental exposure situations, these approaches have not be successful in preventing cancer death in the human population. However, this is not only due to the described 'Pandora's box' situation. At least three other factors are described. Firstly, in the industrial world the medical treatment of cancer in patients occurs with high levels of extremely mutagenic agents. Actually, both in number of persons and in exposure levels such medical treatment is the single largest exposure of humans to known carcinogens. Although such treatments are very effective in curing the tumor as present in the patient, the recurrence of cancer in those patients later in life is very high. In other words: "curing cancer is not the same as preventing cancer death in the human population". Secondly, the rate of cancer death in the human population is also determined by the efficacy in which other major causes of death are prevented. For instance, cardiovascular diseases are the major cause of death in humans in the industrialized world. There is evidence that the treatment of cardiovascular diseases is more successful than that of cancer. On a population level this will result in increase of cancer being the ultimate death cause. Finally, the improvement of medical treatment of diseases together with an improved quality of life will lead to increase average age of the population. Because the onset of most cancer is long after the exposure to carcinogens-in human often more than 30 years-cancer is predominantly a disease of the old age. This means that if the average age of human increases, there will be a selective preference of cancer becoming an even more important cause of death. This especially will be pronounced in those countries were the age distribution in a population is abnormal.

The Drosophila melanogaster DmRAD54 gene plays a crucial role in double-strand break repair after P-element excision and acts synergistically with Ku70 in the repair of X-ray damage.

The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogaster strain deficient in the RAD54 homolog DmRAD54 is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54(-/-) flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, a DmRad54(-/-)/DmKu70(-/-) double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements in Drosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3' overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks.

The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage.

In this study the role of nucleotide excision repair (NER) in protecting mouse embryonic stem (ES) cells against the genotoxic effects of UV-photolesions was analysed. Repair of cyclobutane pyrimidine dimers (CPD) in transcribed genes could not be detected whereas the removal of (6-4) photoproducts (6-4PP) was incomplete, already reaching its maximum (30%) 4 h after irradiation. Measurements of repair replication revealed a saturation of NER activity at UV doses >5 J/m2 while at a lower dose (2.5 J/m2) the repair kinetics were similar to those in murine embryonic fibroblasts (MEFs). Cytotoxic and mutagenic effects of photolesions were determined in ES cells differing in NER activity. ERCC1-deficient ES cells were hypermutable (10-fold) compared to wild-type cells, indicating that at physiologically relevant doses ES cells efficiently remove photolesions. The effect of the NER deficiency on cytoxicity was only 2-fold. Exposure to high UV doses (10 J/m2) resulted in a rapid and massive induction of apoptosis. Possibly, to avoid the accumulation of mutated cells, ES cells rely on the induction of a strong apoptotic response with a simultaneous shutting down of NER activity.