Xpf suppresses the mutagenic consequences of phagocytosis in Dictyostelium.

As time passes, mutations accumulate in the genomes of all living organisms. These changes promote genetic diversity, but also precipitate ageing and the initiation of cancer. Food is a common source of mutagens, but little is known about how nutritional factors cause lasting genetic changes in the consuming organism. Here, we describe an unusual genetic interaction between DNA repair in the unicellular amoeba Dictyostelium discoideum and its natural bacterial food source. We found that Dictyostelium deficient in the DNA repair nuclease Xpf (xpf-) display a severe and specific growth defect when feeding on bacteria. Despite being proficient in the phagocytosis and digestion of bacteria, over time, xpf- Dictyostelium feeding on bacteria cease to grow and in many instances die. The Xpf nuclease activity is required for sustained growth using a bacterial food source. Furthermore, the ingestion of this food source leads to a striking accumulation of mutations in the genome of xpf- Dictyostelium This work therefore establishes Dictyostelium as a model genetic system to dissect nutritional genotoxicity, providing insight into how phagocytosis can induce mutagenesis and compromise survival fitness.

The Fanconi anaemia components UBE2T and FANCM are functionally linked to nucleotide excision repair.

The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes--UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair.

Xpf and not the Fanconi anaemia proteins or Rev3 accounts for the extreme resistance to cisplatin in Dictyostelium discoideum.

Organisms like Dictyostelium discoideum, often referred to as DNA damage "extremophiles", can survive exposure to extremely high doses of radiation and DNA crosslinking agents. These agents form highly toxic DNA crosslinks that cause extensive DNA damage. However, little is known about how Dictyostelium and the other "extremophiles" can tolerate and repair such large numbers of DNA crosslinks. Here we describe a comprehensive genetic analysis of crosslink repair in Dictyostelium discoideum. We analyse three gene groups that are crucial for a replication-coupled repair process that removes DNA crosslinks in higher eukarya: The Fanconi anaemia pathway (FA), translesion synthesis (TLS), and nucleotide excision repair. Gene disruption studies unexpectedly reveal that the FA genes and the TLS enzyme Rev3 play minor roles in tolerance to crosslinks in Dictyostelium. However, disruption of the Xpf nuclease subcomponent results in striking hypersensitivity to crosslinks. Genetic interaction studies reveal that although Xpf functions with FA and TLS gene products, most Xpf mediated repair is independent of these two gene groups. These results suggest that Dictyostelium utilises a distinct Xpf nuclease-mediated repair process to remove crosslinked DNA. Other DNA damage-resistant organisms and chemoresistant cancer cells might adopt a similar strategy to develop resistance to DNA crosslinking agents.

A Dictyostelium homologue of the metazoan Cbl proteins regulates STAT signalling.

Cbl proteins downregulate metazoan signalling pathways by ubiquitylating receptor tyrosine kinases, thereby targeting them for degradation. They contain a phosphotyrosine-binding region, comprising an EF-hand and an SH2 domain, linked to an E3 ubiquitin-ligase domain. CblA, a Dictyostelium homologue of the Cbl proteins, contains all three conserved domains. In a cblA(-) strain early development occurs normally but migrating cblA(-) slugs frequently fragment and the basal disc of the culminants that are formed are absent or much reduced. These are characteristic features of mutants in signalling by DIF-1, the low-molecular-mass prestalk and stalk cell inducer. Tyrosine phosphorylation of STATc is induced by DIF-1 but in the cblA(-) strain this response is attenuated relative to parental cells. We present evidence that CblA fulfils this function, as a positive regulator of STATc tyrosine phosphorylation, by downregulating PTP3, the protein tyrosine phosphatase responsible for dephosphorylating STATc. Thus Cbl proteins have an ancient origin but, whereas metazoan Cbl proteins regulate tyrosine kinases, the Dictyostelium Cbl regulates via a tyrosine phosphatase.

Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase.

STATc becomes tyrosine phosphorylated and accumulates in the nucleus when Dictyostelium cells are exposed to the prestalk cell inducer Differentiation inducing factor 1 (DIF-1), or are subjected to hyper-osmotic stress. We show that the protein tyrosine phosphatase PTP3 interacts directly with STATc and that STATc is refractory to activation in PTP3 overexpressing cells. Conversely, overexpression of a dominant inhibitor of PTP3 leads to constitutive tyrosine phosphorylation and ectopic nuclear localisation of STATc. Treatment of cells with DIF-1 or exposure to hyper-osmotic stress induces a decrease in biochemically assayable PTP3 activity and both agents also induce serine-threonine phosphorylation of PTP3. These observations suggest a novel mode of STAT activation, whereby serine-threonine phosphorylation of a cognate protein tyrosine phosphatase results in the inhibition of its activity, shifting the phosphorylation-dephosphorylation equilibrium in favour of phosphorylation.

Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development.

We describe a rapid method for creating Dictyo stelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topoisomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bs(r)) cassette linked to a bacterial tetracycline resistance (tet(r)) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bs(r)-tet(r) cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictyostelium kinases, GbpC and pats1. Like them it contains a leucine-rich repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures.