GEN1 from a thermophilic fungus is functionally closely similar to non-eukaryotic junction-resolving enzymes

AbstractProcessing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3′ to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1

Dynamics under the Telomeric Bridge

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Structural identity of telomeric complexes

International audienceA major issue in telomere research is to understand how the integrity of chromosome ends is controlled. Although several nucleoprotein complexes have been described at the telomeres of different organisms, it is still unclear how they confer a structural identity to chromosome ends in order to mask them from DNA repair and to ensure their proper replication. In this review, we describe how telomeric nucleoprotein complexes are structured, comparing different organisms and trying to link these structures to telomere biology. It emerges that telomeres are formed by a complex and specific network of interactions between DNA, RNA and proteins. The fact that these interactions and associated activities are reinforcing each other might help to guaranty the robustness of telomeric functions across the cell cycle and in the event of cellular perturbations. We propose that telomeric nucleoprotein complexes orient cell fate through dynamic transitions in their structures and their organizatio

Functional interaction between poly(ADP-Ribose) polymerase 2 (PARP-2) and TRF2: PARP activity negatively regulates TRF2

The DNA damage-dependent poly(ADP-ribose) polymerase-2 (PARP-2) is, together with PARP-1, an active player of the base excision repair process, thus defining its key role in genome surveillance and protection. Telomeres are specialized DNA-protein structures that protect chromosome ends from being recognized and processed as DNA strand breaks. In mammals, telomere protection depends on the T2AG3 repeat binding protein TRF2, which has been shown to remodel telomeres into large duplex loops (t-loops). In this work we show that PARP-2 physically binds to TRF2 with high affinity. The association of both proteins requires the N-terminal domain of PARP-2 and the myb domain of TRF2. Both partners colocalize at promyelocytic leukemia bodies in immortalized telomerase-negative cells. In addition, our data show that PARP activity regulates the DNA binding activity of TRF2 via both a covalent heteromodification of the dimerization domain of TRF2 and a noncovalent binding of poly(ADP-ribose) to the myb domain of TRF2. PARP-2/ primary cells show normal telomere length as well as normal telomerase activity compared to wild-type cells but display a spontaneously increased frequency of chromosome and chromatid breaks and of ends lacking detectable T2AG3 repeats. Altogether, these results suggest a functional role of PARP-2 activity in the maintenance of telomere integrity

Longevity strategies in response to light in the reef coral Stylophora pistillata

International audienceAging is a multifactorial process that results in progressive loss of regenerative capacity and tissue function while simultaneously favoring the development of a large array of age-related diseases. Evidence suggests that the accumulation of senescent cells in tissue promotes both normal and pathological aging. Oxic stress is a key driver of cellular senescence. Because symbiotic long-lived reef corals experience daily hyperoxic and hypoxic transitions, we hypothesized that these long-lived animals have developed specific longevity strategies in response to light. We analyzed transcriptome variation in the reef coral Stylophora pistillata during the day–night cycle and revealed a signature of the FoxO longevity pathway. We confirmed this pathway by immunofluorescence using antibodies against coral FoxO to demonstrate its nuclear translocation. Through qPCR analysis of nycthemeral variations of candidate genes under different light regimens, we found that, among genes that were specifically up- or downregulated upon exposure to light, human orthologs of two “light-up” genes (HEY1 and LONF3) exhibited anti-senescence properties in primary human fibroblasts. Therefore, these genes are interesting candidates for counteracting skin aging. We propose a large screen for other light-up genes and an investigation of the biological response of reef corals to light (e.g., metabolic switching) to elucidate these processes and identify effective interventions for promoting healthy aging in humans