Cupriavidus (Wautersia, Ralstonia, Alcaligenes) metallidurans strain CH34is a well-studied example of a metal-resistant proteobacterium. Genome sequence analysis revealed the presence of a variety of paralogs of proteins that were previously shown to be involved in heavy metal resistance. Which advantage has C. metallidurans in maintaining all these paralogs during evolution? Paralogs investigated belong to the families RND (resistance nodulation cell division) or CHR (chromate resistance). The respective genes were localized by PCR either on one of the two native megaplasmids pMOL28 and pMOL30 of strain CH34, or on its chromosomal DNA. Gene expression was studied by real-time reverse transcriptase PCR and by reporter gene constructs. Genes found to be inducible were disrupted and their contribution to metal resistance measured. When two or three highly related genes were present, usually one was inducible by heavy metals while the other one or two were silent or constitutively expressed. This suggests that C. metallidurans CH34 carries a variety of no longer or not yet used genes that might serve as surplus material for further developments, an advantage that may compensate for the costs of maintaining these genes during evolution.

Regulation of homologous recombination (HR) represents the best-characterized DNA repair function of p53. The role of p53 phosphorylation in DNA repair is largely unknown. Here, we show that wild-type p53 repressed repair of DNA double-strand breaks (DSBs) by HR in a manner partially requiring the ATM/ATR phosphorylation site, serine 15. Cdk-mediated phosphorylation of serine 315 was dispensable for this anti-recombinogenic effect. However, without targeted cleavage of the HR substrate, serine 315 phosphorylation was necessary for the activation of topoisomerase I-dependent HR by p53. Moreover, overexpression of cyclin A1, which mimics the situation in tumors, inappropriately stimulated DSB-induced HR in the presence of oncogenic p53 mutants (not Wtp53). This effect required cyclin A1/cdk-mediated phosphorylation for stable complex formation with topoisomerase I. We conclude that p53 mutants have lost the balance between activation and repression of HR, which results in a net increase of potentially mutagenic DNA rearrangements. Our data provide new insight into the mechanism underlying gain-of-function of mutant p53 in genomic instability.

Resveratrol (RV) inhibits tumour initiation, promotion and progression which has mainly been explained by its properties in cell cycle control and apoptosis induction. So far, ambiguous observations have been published regarding its influence on genomic stability. To study RV's effects on DNA double-strand break (DSB) repair, we applied the established enhanced green fluorescent protein (EGFP)- and I-SceI-based assay system on RV-treated lymphoblastoid cell lines (LCLs). We show that RV inhibits both, homologous recombination (HR) and non-homologous end joining (NHEJ) independently of its known growth and death regulatory functions. Using (i) the isogenic cell lines TK6 and WTK1, which differ in their p53 status, (ii) LCLs from patients with ataxia telangiectasia, (iii) shRNA-mediated p53 knockdown and (iv) chemical inhibition of ATM/ATR by caffeine, we established an ATM-p53-dependent pathway of HR inhibition by RV. Additional use of LCLs from Nijmegen breakage syndrome patients furthermore provided evidence for an ATM/ATR-Nbs1-dependent inhibition of microhomology-mediated NHEJ after RV treatment. We propose that activation of ATM and/or ATR is a central effect of RV. Repression of error-prone recombination subpathways could at least partially explain the chemopreventive effects of this natural plant constituent in animal cancer models.

MLL (myeloid/lymphoid or mixed-lineage leukemia) rearrangements are frequent in therapy-related and childhood acute leukemia, and are associated with poor prognosis. The majority of the rearrangements fall within a 7.3-kb MLL breakpoint cluster region (MLLbcr), particularly in a 0.4-kb hotspot at the intron11-exon12 boundary. The underlying mechanisms are poorly understood, though multiple pathways including early apoptotic signaling, accompanied by high-order DNA fragmentation, have been implicated. We introduced the MLLbcr hotspot in an EGFP-based recombination reporter system and demonstrated enhancement of both spontaneous and genotoxic treatment-induced DNA recombination by the MLLbcr in various human cell types. We identified Endonuclease G (EndoG), an apoptotic nuclease, as an essential factor for MLLbcr-specific DNA recombination after induction of replication stress. We provide evidence for replication stress-induced nuclear accumulation of EndoG, DNA binding by EndoG as well as cleavage of the chromosomal MLLbcr locus in a manner requiring EndoG. We demonstrate additional dependency of MLLbcr breakage on ATM signaling to histone H2B monoubiquitinase RNF20, involved in chromatin relaxation. Altogether our findings provide a novel mechanism underlying MLLbcr destabilization in the cells of origin of leukemogenesis, with replication stress-activated, EndoG-mediated cleavage at the MLLbcr, which may serve resolution of the stalled forks via recombination repair, however, also permits MLL rearrangements.

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ≥90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.