Pol η–dependent DNA synthesis at stalled replication forks during S phase suppresses chronic fragile site instability by preventing checkpoint-blind under-replicated DNA in mitosis.

Chromosomal common fragile sites (CFSs) are unstable genomic regions that break under replication stress and are involved in structural variation. They frequently are sites of chromosomal rearrangements in cancer and of viral integration. However, CFSs are undercharacterized at the molecular level and thus difficult to predict computationally. Newly available genome-wide profiling studies provide us with an unprecedented opportunity to associate CFSs with features of their local genomic contexts. Here, we contrasted the genomic landscape of cytogenetically defined aphidicolin-induced CFSs (aCFSs) to that of nonfragile sites, using multiple logistic regression. We also analyzed aCFS breakage frequencies as a function of their genomic landscape, using standard multiple regression. We show that local genomic features are effective predictors both of regions harboring aCFSs (explaining ∼81% of the deviance in logistic regression models) and of aCFS breakage frequencies (explaining ∼45% of the variance in standard regression models). In our optimal models (based on a combination of biological interpretability and high R-squared value), aCFSs are predominantly located in G-negative chromosomal bands and away from centromeres, are enriched in Alu repeats, and have high DNA flexibility. In alternative models, CpG island density, H3K4me1 coverage, and mononucleotide microsatellite coverage are significant predictors. Also, aCFSs have high fragility when colocated with evolutionarily conserved chromosomal breakpoints. Our models are predictive of the fragility of aCFSs mapped at a higher resolution. Importantly, the genomic features we identified here as significant predictors of fragility allow us to draw valuable inferences on the molecular mechanisms underlying aCFSs.

A tandem repeat’s (TR) propensity to mutate increases with repeat number, and can become very pronounced beyond a critical boundary, transforming it into a microsatellite (MS). However, a clear understanding of the mutational behavior of different TR classes and motifs and related mechanisms is lacking, as is a consensus on the existence of a boundary separating short TRs (STRs) from MSs. This hinders our understanding of MSs’ mutational properties and their effective use as genetic markers. Using indel calls for 179 individuals from 1000 Genomes Pilot-1 Project, we determined polymorphism incidence for four major TR classes, and formalized its varying relationship with repeat number using segmented regression. We observed a biphasic regime with a transition from a faster to a slower exponential growth at 9, 5, 4, and 4 repeats for mono-, di-, tri-, and tetranucleotide TRs, respectively. We used an in vitro mutagenesis assay to evaluate the contribution of strand slippage errors to mutability. STRs and MSs differ in their absolute polymorphism levels, but more importantly in their rates of mutability growth. Although strand slippage is a major factor driving mononucleotide polymorphism incidence, dinucleotide polymorphism incidence is greater than that expected due to strand slippage alone, indicating that additional cellular factors might be driving dinucleotide mutability in the human genome. Leveraging on hundreds of human genomes, we present the first comprehensive, genome-wide analysis of TR mutational behavior, encompassing several motif sizes and compositions.

Common fragile sites (CFS) are hotspots of chromosomal breakage, and CFS breakage models involve perturbations of DNA replication. Here, we analyzed the contribution of specific repetitive DNA sequence elements within CFS to the inhibition of DNA synthesis by replicative and specialized DNA polymerases. The efficiency of in vitro DNA synthesis was quantitated using templates corresponding to regions within FRA16D and FRA3B harboring AT-rich microsatellite and quasi-palindrome (QP) sequences. QPs were predicted to form stems of ~75-100% self homology, separated by 3-9 bases of intervening sequences. Analysis of DNA synthesis progression by human DNA polymerase δ (Pol δ) demonstrated significant synthesis perturbation both at [A]n and [TA]n repeats in a length-dependent manner, and at short (<40 basepairs) QP sequences. DNA synthesis by the Y-family polymerase κ was significantly more efficient than Pol δ through both types of repetitive elements. Using DNA trap experiments, we show that Pol δ pauses within CFS sequences are sites of enzyme dissociation, and dissociation was observed in the presence of RFC-loaded PCNA. We propose that enrichment of microsatellite and QP elements at CFS regions contributes to fragility by perturbing replication through multiple mechanisms, including replicative DNA polymerase pausing and dissociation. Our finding that Pol δ dissociates at specific CFS sequences is significant, since dissociation of the replication machinery and inability to efficiently recover the replication fork can lead to fork collapse and/or formation of double-strand breaks in vivo. Our biochemical studies also extend the potential involvement of Y-family polymerases in CFS maintenance to include polymerase κ.

The integrin α9β1 is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin C and osteopontin. We found that this integrin is a receptor for nerve growth factor (NGF) and two other neurotrophins, brain-derived neurotrophic factor and NT3, using a cell adhesion assay with the α9SW480 cell line. Interaction of α9β1 with NGF was confirmed in an ELISA assay by direct binding to purified integrin. α9β1 integrin binds to neurotrophins in a manner similar to another common neurotrophin receptor, p75NTR (NGFR), although α9β1 activity is correlated with induction of pro-survival and pro-proliferative signaling cascades. This property of α9β1 resembles the interaction of NGF with a high affinity receptor, TrkA, however, this integrin shows a low affinity for NGF. NGF induces chemotaxis of cells expressing α9β1 and their proliferation. Moreover, α9β1 integrin is a signaling receptor for NGF, which activates the MAPK (Erk1/2) pathway. The α9β1-dependent chemotactic ability of NGF appears to result from the activation of paxillin.

Cancer Biology & Therapy 1507 VP12 and viperistatin inhibit melanoma metastasisViperistatin and VP12 isolated from Vipera paleastinae venom showed a potent inhibitory activity against collagen receptors, α1β1 and α2β1 integrins, respectively. Structurally, viperistatin belongs to the disintegrin family of proteins, whereas VP12 is composed of two subunits VP12A and VP12B displaying amino acid sequence homology with heterodimeric C-lectin type proteins. Viperistatin and VP12 used separately and simultaneously inhibited pro-metastatic activities of melanoma cells lines. The level of inhibition of MV3 and HS.939T human cell lines in cell adhesion and migration assays by both compounds was correlated with expression of α1β1 and α2β1 integrins on the cell surface. MV3 cells express collagen receptors to much higher extent than HS.939T and required the application of higher concentrations of inhibitors to block their adhesion to collagen types I and IV. A melanoma cell transmigration assay through a dHMVEC layer revealed that α1β1 integrin plays a significant role in invasion of HS.939T cells, while α2β1 integrin appears to be more important for MV3 cells. In an animal model of hematogenous metastasis of the mouse B16F10 cell line, the inhibitory effect of viperistatin and VP12 was only partial. These data suggest that collagen receptors may be an interesting target for development of new anti-metastatic therapies.

Integrin signaling is comprised of well-characterized pathways generally involved in cell survival. alpha(9)beta(1) integrin has recently become a target of study and has been shown to present pro-survival effects on neutrophils. However, there are no detailed studies on how alpha(9)beta(1) integrin-coupled signaling pathways interact and how they converge to finally modulate spontaneous apoptosis in neutrophils. In this regard we sought to investigate the main signaling events triggered by alpha(9)beta(1) integrin engagement and how these signaling pathways modulate the apoptotic program of human neutrophils. Using VLO5, a snake venom disintegrin shown to bind to alpha(9)beta(1) integrin in neutrophils, we demonstrate that alpha(9)beta(1) integrin engagement leads to the activation of integrin signaling pathways and potently reduces neutrophil spontaneous apoptosis. These effects are dependent on the activation of PI3K and MAPK pathways, since both LY294002 (PI3K inhibitor) or PD95059 (MEK inhibitor) reverted the effects of VLO5/alpha(9)beta(1) interaction. Moreover we show that VLO5/alpha(9)beta(1) engagement induces NF-kappaB nuclear translocation and increases the ratio between anti- and pro-apoptotic proteins by inducing the degradation of pro-apoptotic protein Bad and increasing the expression of anti-apoptotic protein Bcl-x(L). VLO5 also inhibited the early steps of neutrophil spontaneous apoptosis by preventing Bax translocation to the outer mitochondrial membrane and consequent cytochrome c release. In conclusion, as the mechanistic details of alpha(9)beta(1) integrin signaling pathways in human neutrophils becomes clearer, it should become possible to develop new therapeutic agents for human diseases where neutrophils play a prominent role.

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