DNA binding to SMC ATPases-trapped for release.

The SMC/Rad50/RecN proteins are universal DNA­associated ABC­type ATPases with crucial functions in genome maintenance. New insights into Rad50-DNA complex structure and cohesin regulation inspire a speculative look at the entire superfamily. Identification of a continuous DNA binding site across the Rad50 dimer interface (Liu et al, 2016; Seifert et al, 2016) suggests a similar site in cohesin. The localization of this site hints a DNA-activated mechanism for cohesin removal from chromosomes.

The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker.

The structural maintenance of chromosome (SMC) protein complexes cohesin and condensin and the Smc5/6 complex (Smc5/6) are crucial for chromosome dynamics and stability. All contain essential ATPase domains, and cohesin and condensin interact with chromosomes through topological entrapment of DNA. However, how Smc5/6 binds DNA and chromosomes has remained largely unknown. Here, we show that purified Smc5/6 binds DNA through a mechanism that requires ATP hydrolysis by the complex and circular DNA to be established. This also promotes topoisomerase 2-dependent catenation of plasmids, suggesting that Smc5/6 interconnects two DNA molecules using ATP-regulated topological entrapment of DNA, similar to cohesin. We also show that a complex containing an Smc6 mutant that is defective in ATP binding fails to interact with DNA and chromosomes and leads to cell death with concomitant accumulation of DNA damage when overexpressed. Taken together, these results indicate that Smc5/6 executes its cellular functions through ATP-regulated intermolecular DNA linking.

The Drosophila midkine/pleiotrophin homologues Miple1 and Miple2 affect adult lifespan but are dispensable for alk signaling during embryonic gut formation.

Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.

Jeb/Alk signalling regulates the Lame duck GLI family transcription factor in the Drosophila visceral mesoderm.

The Jelly belly (Jeb)/Anaplastic Lymphoma Kinase (Alk) signalling pathway regulates myoblast fusion in the circular visceral mesoderm (VM) of Drosophila embryos via specification of founder cells. However, only a limited number of target molecules for this pathway are described. We have investigated the role of the Lame Duck (Lmd) transcription factor in VM development in relationship to Jeb/Alk signal transduction. We show that Alk signalling negatively regulates Lmd activity post-transcriptionally through the MEK/MAPK (ERK) cascade resulting in a relocalisation of Lmd protein from the nucleus to cytoplasm. It has previously been shown that downregulation of Lmd protein is necessary for the correct specification of founder cells. In the visceral mesoderm of lmd mutant embryos, fusion-competent myoblasts seem to be converted to 'founder-like' cells that are still able to build a gut musculature even in the absence of fusion. The ability of Alk signalling to downregulate Lmd protein requires the N-terminal 140 amino acids, as a Lmd(141-866) mutant remains nuclear in the presence of active ALK and is able to drive robust expression of the Lmd downstream target Vrp1 in the developing VM. Our results suggest that Lmd is a target of Jeb/Alk signalling in the VM of Drosophila embryos.

The SMC complexes, DNA and chromosome topology: right or knot?

Topology is the study of geometric properties that are preserved during bending, twisting and stretching of objects. In the context of the genome, topology is discussed at two interconnected and overlapping levels. The first focuses the DNA double helix itself, and includes alterations such as those triggered by DNA interacting proteins, processes which require the separation of the two DNA strands and DNA knotting. The second level is centered on the higher order organization of DNA into chromosomes, as well as dynamic conformational changes that occur on a chromosomal scale. Here, we refer to the first level as "DNA topology", the second as "chromosome topology". Since their identification, evidences suggesting that the so called structural maintenance of chromosomes (SMC) protein complexes are central to the interplay between DNA and chromosome topology have accumulated. The SMC complexes regulate replication, segregation, repair and transcription, all processes which influence, and are influenced by, DNA and chromosome topology. This review focuses on the details of the relationship between the SMC complexes and topology. It also discusses the possibility that the SMC complexes are united by a capability to sense the geometrical chirality of DNA crossings.

Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival.

The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell survival, MAPK p38 phosphorylation, RhoA activation and actin cytoskeleton reorganization in response to DNA damage. Our data reveal a previously unrecognized crosstalk between DNA damage and cytoskeleton dynamics in the regulation of cell survival, and might provide new insights on the role of chronic bacteria infection in carcinogenesis.

Midkine and pleiotrophin have bactericidal properties: preserved antibacterial activity in a family of heparin-binding growth factors during evolution.

Antibacterial peptides of the innate immune system combat pathogenic microbes, but often have additional roles in promoting inflammation and as growth factors during tissue repair. Midkine (MK) and pleiotrophin (PTN) are the only two members of a family of heparin-binding growth factors. They show restricted expression during embryogenesis and are up-regulated in neoplasia. In addition, MK shows constitutive and inflammation-dependent expression in some non-transformed tissues of the adult. In the present study, we show that both MK and PTN display strong antibacterial activity, present at physiological salt concentrations. Electron microscopy of bacteria and experiments using artificial lipid bilayers suggest that MK and PTN exert their antibacterial action via a membrane disruption mechanism. The predicted structure of PTN, employing the previously solved MK structure as a template, indicates that both molecules consist of two domains, each containing three antiparallel beta-sheets. The antibacterial activity was mapped to the unordered C-terminal tails of both molecules and the last beta-sheets of the N-terminals. Analysis of the highly conserved MK and PTN orthologues from the amphibian Xenopus laevis and the fish Danio rerio suggests that they also harbor antibacterial activity in the corresponding domains. In support of an evolutionary conserved function it was found that the more distant orthologue, insect Miple2 from Drosophila melanogaster, also displays strong antibacterial activity. Taken together, the findings suggest that MK and PTN, in addition to their earlier described activities, may have previously unrealized important roles as innate antibiotics.

Chromosome segregation and double-strand break repair - a complex connection.

Genome stability requires correct chromosome segregation and DNA repair. Failure of these processes leads to cell death or accumulation of chromosomal aberrations, as often observed in tumor cells. An increasing number of observations indicate that segregation and DNA double-strand break (DSB) repair are functionally connected by the Cohesin and Smc5/6 protein complexes. Through their interaction with the duplicated genome, these complexes play essential roles in both chromosome segregation and repair by sister chromatid recombination. Both are also recruited to DSBs, and their chromosomal association is similarly regulated. Interestingly, recent studies of Cohesin suggest that DSB formation could promote proper mitotic chromosome segregation. This is reminiscent of segregation in meiotic cells, which is facilitated by break-induced chromosomal tethering.