Disparate contributions of the Fanconi anemia pathway and homologous recombination in preventing spontaneous mutagenesis

Fanconi anemia (FA) is a chromosomal instability disorder in which DNA-damage processing defects are reported for translesion synthesis (TLS), non-homologous end joining (NHEJ) and homologous recombination (HR; both increased and decreased). To reconcile these diverse findings, we compared spontaneous mutagenesis in FA and HR mutants of hamster CHO cells. In the fancg mutant we find a reduced mutation rate accompanied by an increased proportion of deletions within the hprt gene. Moreover, in fancg cells gene amplification at the CAD and dhfr loci is elevated, another manifestation of inappropriate processing of damage during DNA replication. In contrast, the rad51d HR mutant has a greatly elevated rate of hprt mutations, >85% of which are deletions. Our analysis supports the concept that HR faithfully restores broken replication forks, whereas the FA pathway acts more globally to ensure chromosome stability by promoting efficient end joining of replication-derived breaks, as well as TLS and HR

Qualitatively and quantitatively similar effects of active and passive maternal tobacco smoke exposure on in utero mutagenesis at the HPRT locus

BACKGROUND: Induced mutagenesis in utero is likely to have life-long repercussions for the exposed fetus, affecting survival, birth weight and susceptibility to both childhood and adult-onset diseases, such as cancer. In the general population, such exposures are likely to be a consequence of the lifestyle choices of the parents, with exposure to tobacco smoke one of the most pervasive and easily documented. Previous studies attempting to establish a direct link between active smoking and levels of somatic mutation have largely discounted the effects of passive or secondary exposure, and have produced contradictory results. METHODS: Data from three studies of possible smoking effects on in utero mutagenesis at the HPRT locus were compiled and reanalyzed, alone and in combination. Where possible, passive exposure to environmental tobacco smoke was considered as a separate category of exposure, rather than being included in the non-smoking controls. Molecular spectra from these studies were reanalyzed after adjustment for reported mutation frequencies from the individual studies and the entire data set. RESULTS: A series of related studies on mutation at the X-linked HPRT locus in human newborn cord blood samples has led to the novel conclusion that only passive maternal exposure to tobacco mutagens has a significant effect on the developing baby. We performed a pooled analysis of the complete data from these studies, at the levels of both induced mutation frequency and the resulting mutational spectrum. CONCLUSION: Our analysis reveals a more commonsensical, yet no less cautionary result: both active maternal smoking and secondary maternal exposure produce quantitatively and qualitatively indistinguishable increases in fetal HPRT mutation. Further, it appears that this effect is not perceptibly ameliorated if the mother adjusts her behavior (i.e. stops smoking) when pregnancy is confirmed, although this conclusion may also be affected by continued passive exposure

Differences in methylation patterns of the alpha-fetoprotein and albumin genes in hepatic and non hepatic developing rat tissues.

By use of different restriction enzymes sensitive to internal cytosine methylation (HpaII, AvaI, HhaI) we have analysed the methylation patterns of albumin and AFP genes in tissues and cell lines with high (liver, yolk sac, hepatoma cell lines), low (fetal and neonatal kidney) or undetectable (spleen, JF1 fibroblasts) expression of either gene. We show that expression of the AFP gene is associated to the demethylation of a whole region or domain extending from -4 to +3 Kb. Moreover, demethylation of a site located at the upstream limit of this domain appears to be correlated with the commitment of the cell type to synthesize AFP. As concerns the albumin gene, we show that the domain in which demethylation is correlated with active gene transcription in hepatoma cell lines has different borders than in tissue. This difference might be related to the different amounts of mRNA synthesized or to an alteration in gene regulation in tumor cells. Finally, we show that low expression of albumin and AFP genes in fetal and neonatal kidney is not correlated with domain demethylation, suggesting that the regulatory mechanisms of expression of these genes are different in kidney as compared with liver