Human CST promotes telomere duplex replication and general replication restart after fork stalling

Mammalian CST (CTC1-STN1-TEN1) associates with telomeres and depletion of CTC1 or STN1 causes telomere defects. However, the function of mammalian CST remains poorly understood. We show here that depletion of CST subunits leads to both telomeric and non-telomeric phenotypes associated with DNA replication defects. Stable knockdown of CTC1 or STN1 increases the incidence of anaphase bridges and multi-telomeric signals, indicating genomic and telomeric instability. STN1 knockdown also delays replication through the telomere indicating a role in replication fork passage through this natural barrier. Furthermore, we find that STN1 plays a novel role in genome-wide replication restart after hydroxyurea (HU)-induced replication fork stalling. STN1 depletion leads to reduced EdU incorporation after HU release. However, most forks rapidly resume replication, indicating replisome integrity is largely intact and STN1 depletion has little effect on fork restart. Instead, STN1 depletion leads to a decrease in new origin firing. Our findings suggest that CST rescues stalled replication forks during conditions of replication stress, such as those found at natural replication barriers, likely by facilitating dormant origin firing

A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes

International audienceHuman E-cadherin promotes entry of the bacterial pathogen Listeria monocytogenes into mammalian cells by interacting with internalin (InlA), a bacterial surface protein. Here we show that mouse E-cadherin, although very similar to human E-cadherin (85% identity), is not a receptor for internalin. By a series of domain-swapping and mutagenesis experiments, we identify Pro16 of E-cadherin as a residue critical for specificity: a Pro→Glu substitution in human E-cadherin totally abrogates interaction, whereas a Glu→Pro substitution in mouse E-cadherin results in a complete gain of function. A correlation between cell permissivity and the nature of residue 16 in E-cadherins from several species is established. The location of this key specificity residue in a region of E-cadherin not involved in cell-cell adhesion and the stringency of the interaction demonstrated here have important consequences not only for the understanding of internalin function but also for the choice of the animal model to be used to study human listeriosis: mouse, albeit previously widely used, and rat appear as inappropriate animal models to study all aspects of human listeriosis, as opposed to guinea-pig, which now stands as a small animal of choice for future in vivo studies

STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

<div><p>Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. <i>In vivo</i>, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. <i>In vitro</i> binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the <i>in vivo</i> separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The <i>in vitro</i> studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress.</p></div

STN1-OBM rescues viability and restores origin firing after HU treatment.

<p>(A) MTT assay showing viability after HU treatment. Cells were treated with 2 mM HU for the indicated times and harvested for MTT assay 24 hrs later. Values are relative to untreated cells of the same cell type. Each time point was assayed in triplicate and the data are shown as the mean ± S.D from 3 independent experiments. For each cell line, the value of the untreated sample was set at 1. (B-D) DNA fiber analysis of origin firing following release from 2 mM HU. (B) Left: schematic showing timing of IdU and CldU labeling relative to HU treatment. Right: types of replication event scored. (C) Representative images of DNA tracks. Red, IdU; Green, CldU. (D) Graph indicating the percent of DNA tracks showing new origin firing (green-only tracks) (n = 7 experiments, mean ± S.E.M, p-values are indicated above bars).</p

STN1-OBM is competent for C-strand fill-in and TPP1 and pol α interaction.

<p>(A-C) Analysis of C-strand fill-in. (A) FACS analysis of DNA content showing synchrony of STN1-OBM cells used in (B). (B) Representative gels showing in-gel hybridization of (TA₂C₃)₄ probe to DNA from cells harvested at the indicated times after release from G1/S block. (C) Quantification of G-overhang abundance. Cell types were analyzed in pairs, n = 3 experiments for shSTN1 + STN1-OBM, mean ± S.E.M.; n = 2 experiments for STN1-OBM + STN1-Res, error bars show min/max values). (D) Western blot showing co-immunoprecipitation of TPP1 with STN1 or STN1-OBM. Cells were transfected with FLAG-STN1 or FLAG-STN1-OBM plus HA-mCherry-TPP1 expression constructs. TPP1 was precipitated with antibody to HA. (E) Co-immunoprecipitation of DNA pol α with CST. Cells were transfected with FLAG-STN1 or FLAG-STN1-OBM, Myc-CTC1 and TEN1. CST was precipitated with FLAG beads.</p

Sequence of oligonucleotides used in DNA binding assays.

<p>Sequence of oligonucleotides used in DNA binding assays.</p

<i>In vivo</i> expression of STN1-OBM causes anaphase bridges.

<p>(A) Western blot showing levels of STN1 in HeLa cells expressing non-target shRNA (shNT) or STN1 shRNA (shSTN1) and shSTN1 cells with sh-resistant mutant STN1 (STN1-OBM) or wild type STN1 (STN1-Res). Blot was probed with antibody to STN1 or to actinin for a loading control. (B) Left; representative images of DAPI-stained anaphase cells with/ without bridges. Anaphase cells with no bridge in shNT (top) and with bridges in shSTN1 and STN1-OBM cells (middle and bottom). Right; quantification of bridges (200 anaphases counted per cell line per experiment. n = 3 experiments, mean ± S.E.M, p-values are indicated above bars).</p

Photo-crosslinking of CST subunits to thiothymidine substituted ssDNA.

<p>(A) Positions of s⁴T substitutions in TelG-18 and TelG-48 DNA substrates. (B) EMSA showing the s⁴T substitutions do not affect CST(WT) or CST(STN1-OBM) binding to TelG-18 or TelG-48. (C-D) Products obtained after photo-crosslinking. Left and right panels: Phosphorimager scans showing ³²P-labeled cross-linking products. Central panels: Western blots from the same gels showing positions of uncross-linked CTC1, STN1 and TEN1. (C) Products obtained with 3’ (left) or 5’ (right) modified TelG-18. (D) Products obtained with 3’ (left) or 5’ (right) modified TelG-48. * indicates cross-linking products observed only in some experiments. They may represent CTC1 or STN1 degradation products. Markers on the phosphorimager scans were obtained by laying the gels on nitrocellulose membrane and marking the positions of the marker bands with radioactive ink. For the Western blots, the membrane was cut into pieces, probed with antibody to CTC1, STN1 or TEN1, reassembled and exposed to film. The film was laid over the membrane and photographed to visualize both the markers and the CST bands. (E) Dynamic binding model of CST showing micro-dissociation of an individual OB fold (blue) to allow binding of an alternative protein (yellow).</p

Analysis of CST DNA binding parameters.

<p>(A) Binding isotherms used to determine apparent dissociation constants for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates. Data were obtained by filter binding assay and fit to a one site specific binding model. Mean ± SEM, n = 3 independent experiments each with a different protein preparation. (B) Dissociation kinetics for CST bound to the indicated substrates The fraction of labeled DNA remaining bound was determined by filter binding at the indicated times. Data were fit to a one phase exponential decay equation to obtain the dissociation rate (t½)). Mean ± SEM, n = 3 independent experiments. (C) Table summarizing Kd(app) and t½ for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates.—undetectable binding, ND: not determined.</p