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.

Molecular cloning, expression and chromosomal localisation of the mouse Rev3l gene, encoding the catalytic subunit of polymerase zeta.

The REV3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta which is involved in translesion synthesis. The mouse homolog of this gene, Rev3l, was cloned and sequenced. The gene encodes a putative protein of 3122 amino acids. The sequence conservation to its yeast counterpart is restricted to several regions. In the carboxy-terminal part of the protein all six domains are present that are characteristic for alpha-type DNA polymerases. In the amino-terminal part of the protein two regions can be identified with considerable similarity to the NT boxes of mouse polymerase delta. In addition, a region of 60 residues unique for the REV3 homologs can be found in the middle part of the protein. The mouse REV3L protein shows strong sequence conservation with the recently cloned human REV3L protein (86% identity overall). Northern blot analysis of various tissues of the mouse revealed that transcription of the Rev3l gene was highest in brain, ovaries and testis. The human REV3L gene was localised to the long arm of chromosome 6, region 21-22. The mouse equivalent maps to chromosome 10, distal to the c-myb gene, close to the Macs gene.

Carcinogen-induced loss of heterozygosity at the Aprt locus in somatic cells of the mouse.

Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase (Aprt) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH in vivo. After 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal Aprt gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase (Hprt) gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted Aprt allele was lost in 70% of the DMBA-induced Aprt mutants. Fluorescence in situ hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at Aprt. These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss and duplication but not deletion. However, after treatment with the alkylating agent N-ethyl-N-nitrosourea, Aprt had a similar mutagenic response to Hprt while the majority (90%) of N-ethyl-N-nitrosourea-induced Aprt mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at Hprt but not at Aprt. This in vivo study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival and may represent an initiating event in tumorigenesis.

Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation.

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52(-/-) ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.

Determination of spontaneous loss of heterozygosity mutations in Aprt heterozygous mice.

A mouse model was generated to investigate loss of heterozygosity (LOH) events in somatic cells. The adenine phosphoribosyltransferase ( Aprt ) gene was disrupted in embryonic stem cells using a conventional gene targeting approach and subsequently Aprt hetero-zygous and homozygous mice were derived. Aprt homozygous deficient animals were viable though the mendelian inheritance pattern was skewed. On average these mice died at 6 months of age from severe renal failure. In T-lymphocytes of Aprt heterozygous mice the mean spontaneous mutant frequency at the Aprt locus was 8.7 x 10(-6) while the frequency was 0.8 x 10(-6) at the hypoxanthine phosphoribosyltransferase locus. In order to determine whether LOH events contribute to the high spontaneous mutant frequency at the Aprt locus, 140 Aprt mutant T-lymphocyte clones were expanded and analysed by allele-specific PCR. In 97 (69%) of these clones the wild-type allele had been lost. Nine of the mutant clones were characterized in more detail using dual-coloured fluorescence in situ hybridization analysis. Five out of six of the mutant clones which arose from an LOH event, based on the PCR assay, contained a duplication of the targeted allele. Therefore, mitotic recombination or chromosome loss followed by duplication of the remaining homologue appears to be the predominant mechanism for the in vivo generation of Aprt mutant T-lymphocytes.

The design of a new mutation model for active genes: expression of the Escherichia coli lac operon in mammalian cells.

The design of a novel transgenic mouse model is described that should allow analysis of mutations at a single cell level in all tissues of a model animal. The model is based on the correct regulation of the Escherichia coli lac operon in mammalian cells. Induction of a mutation in the lacI gene will result in the loss of transcriptional repression of the lacZ gene in mutated cells. Expression of beta-galactosidase can subsequently be detected at the single cell level. The model was first tested in vitro using transfection of mouse LTK- cells. LacZ expression was very heterogeneous in most of the stable transfectants and seemed to be subject to epigenetic inactivation. One clone (IIB1) was isolated that stably expressed lacZ in more than 99% of its cells. Subsequent introduction of the lacI gene into IIB1 cells resulted in correct transcriptional repression of the lacZ gene that could be alleviated by IPTG, an allosteric inducer of lacI repression. However, in time the extent of beta-galactosidase induction gradually declined suggesting that the prolonged repressed transcriptional state triggers epigenetic inactivation. Variegated expression of the lacZ gene was not confined to cultured cells since several transgenic lines also did not express the lacZ transgene. This study shows that while the susceptibility of the lacZ gene to inactivation processes poses a fundamental problem, correct regulation of the expression of a reporter gene by the lacI repressor protein is feasible in mammalian cells when assayed at the single cell level. Thus, the model can in principle be used for the detection of mutagenic events at the lacI locus. Targeting of the lacZ gene to an endogenous housekeeping gene might prevent epigenetic inactivation. Alternatively, with the use of another reporter gene in the mutation detection system the proposed transgenic mouse model could be realized.