Dpb11, the budding yeast homolog of TopBP1, functions with the checkpoint clamp in recombination repair

Dpb11 is required for the loading of DNA polymerases α and ɛ on to DNA in chromosomal DNA replication and interacts with the DNA damage checkpoint protein Ddc1 in Saccharomyces cerevisiae. The interaction between the homologs of Dpb11 and Ddc1 in human cells and fission yeast is thought to reflect their involvement in the checkpoint response. Here we show that dpb11-1 cells, carrying a mutated Dpb11 that cannot interact with Ddc1, are defective in the repair of methyl methanesulfonate (MMS)-induced DNA damage but not in the DNA damage checkpoint at the permissive temperature. Epistatic analyses suggested that Dpb11 is involved in the Rad51/Rad52-dependent recombination pathway. Ddc1 as well as Dpb11 were required for homologous recombination induced by MMS. Moreover, we found the in vivo association of Dpb11 and Ddc1 with not only the HO-induced double-strand break (DSB) site at MAT locus but also the donor sequence HML during homologous recombination between MAT and HML. Rad51 was required for their association with the HML donor locus, but not with DSB site at the MAT locus. In addition, the association of Dpb11 with the MAT and HML locus after induction of HO-induced DSB was dependent on Ddc1. These results indicate that, besides the involvement in the replication and checkpoint, Dpb11 functions with Ddc1 in the recombination repair process itself

Nuclear β-catenin and CD44 upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells

<p>Abstract</p> <p>Background</p> <p>In breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44⁺/CD24^-stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44⁺/CD24^-subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression?</p> <p>Methods</p> <p>Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment.</p> <p>Results</p> <p>MCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, β-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (<it>PIK3R1</it>, <it>SOCS2</it>, <it>BMP7</it>, <it>CD44 </it>and <it>CD24</it>). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells.</p> <p>Conclusions</p> <p>MCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.</p

Arp4p and Arp4 containing complexes associate with the centromere and telomere

<p><b>Copyright information:</b></p><p>Taken from "Actin-related protein Arp4 functions in kinetochore assembly"</p><p></p><p>Nucleic Acids Research 2007;35(9):3109-3117.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888834.</p><p>© 2007 The Author(s)</p> Cells with 13Myc-tagged Arp4 (YHO312) were grown in YPAD at 20°C for 3 h with 100 ng/ml α-factor. Cells were released by washing in YPAD and incubated in fresh YPAD medium at 20°C. Samples were taken at the time points indicated and analyzed by flow cytometry () and ChIP (). () Flow cytometry analysis of cell cycle profiles. () Input DNA and DNA coimmunoprecipitated with the anti-Myc antibody (IP) were amplified with primer sets corresponding to sequences around centromeres ( and ), the inner region of a large ORF (), a telomere (), and a sub-telomeric region (). To ensure the linearity of the PCR signal, appropriate dilutions of IP samples were used in PCR amplifications. ChIP PCR products were separated by agarose gel electrophoresis. Representative data are shown. () Arp4p, Ino80p, Esa1p and Swr1p interact with and localize to , , , but not to and a subtelomeric region. Flag-tagged Arp4 (YHO311), Ino80 (YHO313), Esa1 (YHO314), Swr1 (YHO315) or untagged (YK402) cells were arrested in G2/M by treatment with nocodazole at 30°C. Cells were fixed with 1% formaldehyde for 15 min and subjected to ChIP. Input DNA and DNA coimmunoprecipitated with the anti-FLAG antibody (IP) were amplified with primer sets corresponding to sequences around , , , , and a subtelomeric region. The templates used were total chromatin (Input) or immunoprecipitate (IP)

The centromeric binding of kinetochore components is partially impaired in mutants

<p><b>Copyright information:</b></p><p>Taken from "Actin-related protein Arp4 functions in kinetochore assembly"</p><p></p><p>Nucleic Acids Research 2007;35(9):3109-3117.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888834.</p><p>© 2007 The Author(s)</p> 3HA-tagged or untagged wild-type and cells were grown in YPAD at 23°C for 3 h with 15 μg/ml nocodazole to ensure that both populations had an equivalent cell cycle distribution since a higher proportion of cells are in G2/M phase. The culture was shifted to 37°C and incubated in the presence of nocodazole for 1 h. Cells were fixed with 1% formaldehyde for 15 min and subjected to ChIP. Input DNA and DNA coimmunoprecipitated with the anti-HA antibody (IP) were amplified with primer sets corresponding to sequences around centromeres (). Quantitative data were obtained by real-time PCR. To ensure the linearity of the PCR signal, appropriate dilutions of IP samples were used in PCR amplifications. In each case, ChIP enrichment is expressed relative to that for a subtelomeric region of chromosome V (9716–9823). Results are expressed as the mean and SD of two independent ChIP experiments. Dashed lines indicate the background level of ChIP signal intensity in an untagged strain. () Schematic of kinetochore components. () The centromere-specific histone H3 variant Cse4p (wild-type cells: YHO805; cells: YHO825), a representative protein of the inner kinetochore Mif2p (wild-type cells: YHO806; cells: YHO826) and Ndc10p (wild-type cells: YHO807; cells: YHO827) were analyzed by ChIP at . () A representative protein of the outer kinetochore Mtw1p (wild-type cells: YHO808; cells: YHO828), Nuf2p (wild-type cells: YHO809; cells: YHO829) and Ctf3p (wild-type cells: YHO810; cells: YHO830) were analysed by ChIP at . () The cohesin component Scc1p (wild-type cells: YHO811; cells: YHO831) was analysed by ChIP at