Running title: Maximal loading of MCM2/4 in late G1

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2-7 complex onto chromatin during G1 phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G1 phase. The immobile fraction of MCM2 and MCM4 increases during G1 phase, suggestive of reiterative licensing. In late G1 phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G1 phase progresses and do not colocalize with sites of DNA synthesis in S phase.Fil: Symeonidou, Ioanna Eleni. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Kotsantis, Panagiotis. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Roukos, Vassilis. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Rapsomaniki, Maria Anna. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Grecco, Hernan Edgardo. Max Planck Institute of Molecular Physiology. Department of Systemic Cell Biology; Alemania; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; Argentina;Fil: Bastiaens, Philippe. Max Planck Institute of Molecular Physiology. Department of Systemic Cell Biology; Alemania;Fil: Taraviras, Stavros. University of Patras. School of Medicine. Laboratory of Physiology; Grecia;Fil: Lygerou, Zoi. University of Patras. School of Medicine. Laboratory of General Biology; Grecia

PARP1 proximity proteomics reveals interaction partners at stressed replication forks

PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1

Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B

Cdt1 Is Differentially Targeted for Degradation by Anticancer Chemotherapeutic Drugs

Background: Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent rereplication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis. Methodology/Principal Finding: In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA. Conclusion/Significance: Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficac

Treatment with 5-Fluoruracil (5-FU) doesn't alter Cdt1 protein expression levels in HeLa or HepG2 cells.

<p>Asynchronous HeLa (A) and HepG2 cells (C) were incubated with 5-FU (0.1 and 100 µg/ml) in the presence of BrdU (20 µM, for 1 h). Cells were subjected to immunofluorescence using antibodies against Cdt1, Cyclin A and BrdU. DNA was visualized with DAPI or Hoechst 3258. The percentage of HeLa (B) and HepG2 (D) cells expressing Cdt1, Cyclin A and BrdU in presence of 5-FU, 0.1 µg/ml (grey columns), 100 µg/ml (black columns) and control cells (white columns) is shown; Data are the mean values of the quantifications from at least 3 different experiments from each condition and represent mean ± SD. **p<0.01, ***p<0.001. (E) HeLa and HepG2 cells were synchronized with nocodazole, released to enter G1 phase, and incubated with 5-FU (10 and 100 µg/ml) for 6 hours. Total cell lysates were extracted and subjected to Western blot analysis using antibodies against Cdt1 and Tubulin. Scale bars: A, C, 50 µm.</p

Differential regulation of Cdt1 in response to the topoisomerase inhibitors Doxorubicin and Etoposide.

<p>HeLa and HepG2 cells were treated for 6 h with (A) Doxorubicin (0.2, 2 and 10 µM) (Doxo) or (B) Etoposide (20 and 80 µM) (Etopo), in the presence or absence of the proteasome inhibitor MG-132 (+MG-132). Total protein extracts were prepared and subjected to western blot analysis using antibodies against Cdt1, PARP, Geminin and Tubulin. (C) HeLa and HepG2 cells were synchronized in M phase with nocodazole, and subsequently were incubated with Etoposide (20 and 80 µM) (lanes 2–3, 7–8) or Doxorubicin (0.2 and 2 µM) (lanes 4–5, 9–10). Protein extracts were subjected to Western blot analysis using antibodies against Cdt1 and Tubulin.</p

Treatment with Tamoxifen does not affect Cdt1 protein expression levels.

<p>HeLa and HepG2 cells were treated with Tamoxifen (0.2, 2 and 10 µM) for 6 h, in absence (lanes 1–4, 9–11) or in presence (lanes 5–8, 12–14) of MG-132. Cells were harvested, protein extracts were prepared and subjected to Western blot analysis using antibodies against Cdt1 and Tubulin as a loading control.</p