MCM9 Is Required for Mammalian DNA Mismatch Repair.

DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects DNA polymerase errors during replication to maintain genomic integrity. In E. coli, the DNA helicase UvrD is implicated in MMR, yet an analogous helicase activity has not been identified in eukaryotes. Here, we show that mammalian MCM9, a protein involved in replication and homologous recombination, forms a complex with MMR initiation proteins (MSH2, MSH3, MLH1, PMS1, and the clamp loader RFC) and is essential for MMR. Mcm9-/- cells display microsatellite instability and MMR deficiency. The MCM9 complex has a helicase activity that is required for efficient MMR since wild-type but not helicase-dead MCM9 restores MMR activity in Mcm9-/- cells. Moreover, MCM9 loading onto chromatin is MSH2-dependent, and in turn MCM9 stimulates the recruitment of MLH1 to chromatin. Our results reveal a role for MCM9 and its helicase activity in mammalian MMR.

Extracting order from heterogeneity: A report on the EpiGeneSys workshop "Single Cell Epigenetics" in Montpellier, June 11-12, 2015.

Understanding epigenetic modifications to chromatin that regulate gene expression and cell-fate decisions is now possible in single cells thanks to recent technological advances. As interdisciplinary approaches are required to derive biological principles, this workshop brought together some of Europe's leading researchers in single-cell epigenetics to share technologies and biological insights.

Studying Translesion DNA Synthesis Using Xenopus In Vitro Systems.

Cell-free extracts derived from Xenopus eggs have been widely used to decipher molecular pathways involved in several cellular processes including DNA synthesis, the DNA damage response, and genome integrity maintenance. We set out assays using Xenopus cell-free extracts to study translesion DNA synthesis (TLS), a branch of the DNA damage tolerance pathway that allows replication of damaged DNA. Using this system, we were able to recapitulate TLS activities that occur naturally in vivo during early embryogenesis. This chapter describes protocols to detect chromatin-bound TLS factors by western blotting and immunofluorescence microscopy upon induction of DNA damage by UV irradiation, monitor TLS-dependent mutagenesis, and perform proteomic screening.

Microtubule assembly by the Apc protein is regulated by importin-beta--RanGTP.

Mutations in the tumour suppressor Adenomatous polyposis coli (Apc) initiate most sporadic colorectal cancers. Apc is implicated in regulating microtubule (MT) dynamics in interphase and mitosis. However, little is known about the underlying mechanism or regulation of this Apc function. We identified importin-beta as a binding partner of Apc that regulates its effect on MTs. Apc binds importin-beta in vitro and in Xenopus egg extracts, and RanGTP inhibits this interaction. The armadillo-like repeat domain of importin-beta binds to the middle of Apc, where it can compete with beta-catenin. In addition, two independent sites in the C terminus of Apc bind the N-terminal region of importin-beta. Binding to importin-beta reduces the ability of Apc to assemble and bundle MTs in vitro and to promote assembly of microtubule asters in Xenopus egg extracts, but does not affect the binding of Apc to MTs or to EB1. Depletion of Apc decreases the formation of cold-stable spindles in Xenopus egg extracts. Importantly, the ability of purified Apc to rescue this phenotype was reduced when it was constitutively bound to importin-beta. Thus, importin-beta binds to Apc and negatively regulates the MT-assembly and spindle-promoting activity of Apc in a Ran-regulatable manner.

BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.

Dynamic localisation of Ran GTPase during the cell cycle.

BACKGROUND: Ran GTPase has multiple functions during the cell division cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. The activity of Ran is determined by both its guanine nucleotide-bound state and its subcellular localization. RESULTS: Here, we have characterised the localisation and mobility of Ran coupled to green fluorescent protein (GFP) during the cell cycle in live human cells. Ran-GFP is nuclear during interphase and is dispersed throughout the cell during mitosis. GFP-RanQ69L, a mutant locked in the GTP-bound state, is less highly concentrated in the nucleus and associates with nuclear pore complexes within the nuclear envelope. During mitosis, GFP-RanQ69L is excluded from chromosomes and localizes to the spindle. By contrast, GFP-RanT24N, a mutant with low affinity for nucleotides, interacts relatively stably with chromatin throughout the cell cycle and is highly concentrated on mitotic chromosomes. CONCLUSION: These results show that Ran interacts dynamically with chromatin, nuclear pore complexes and the mitotic spindle during the cell cycle. These interactions are dependent on the nucleotide-bound state of the protein. Our data indicate that Ran-GTP generated at chromatin is highly mobile and interacts dynamically with distal structures that are involved in nuclear transport and mitotic spindle assembly.

Preventing carryover of peptides and proteins in nano LC-MS separations.

Sample carryover is a significant problem that occurs in high-performance liquid chromatography (HPLC) analysis. Carryover effects cannot be tolerated in any high-performance liquid chromatography-mass spectroscopy (HPLC-MS) separation system, and proteomics analysis must be performed in a separation system with virtually no carryover. Several procedures have been tested for effective and fast removal of interfering peptides and proteins originating from previous analyses in the HPLC system. We have developed and optimized a cleaning method for eliminating carryover caused by the autosampler and the trap column. The new washing method uses an injection of trifluoroethanol into the injection path and onto the trap column to remove strongly bound peptides and proteins, and it includes trifluoroethanol as an additional solvent in the chromatographic mobile phase for enhanced cleaning of the separation column. By application of this method, a significant reduction in carryover was achieved without any loss in the amount of proteins and peptides identified by MS.

Role of importin-beta in the control of nuclear envelope assembly by Ran.

Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin. Here, we show that importin-beta, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-beta on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-beta in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-alpha is functional. Thus, importin-beta functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-alpha with karyophilic proteins carrying classical nuclear localization signals. Importin-beta links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division.

Phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis.

The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.

Regulation of Cdc2/cyclin B activation in Xenopus egg extracts via inhibitory phosphorylation of Cdc25C phosphatase by Ca(2+)/calmodulin-dependent protein [corrected] kinase II.

Activation of Cdc2/cyclin B kinase and entry into mitosis requires dephosphorylation of inhibitory sites on Cdc2 by Cdc25 phosphatase. In vertebrates, Cdc25C is inhibited by phosphorylation at a single site targeted by the checkpoint kinases Chk1 and Cds1/Chk2 in response to DNA damage or replication arrest. In Xenopus early embryos, the inhibitory site on Cdc25C (S287) is also phosphorylated by a distinct protein kinase that may determine the intrinsic timing of the cell cycle. We show that S287-kinase activity is repressed in extracts of unfertilized Xenopus eggs arrested in M phase but is rapidly stimulated upon release into interphase by addition of Ca2+, which mimics fertilization. S287-kinase activity is not dependent on cyclin B degradation or inactivation of Cdc2/cyclin B kinase, indicating a direct mechanism of activation by Ca2+. Indeed, inhibitor studies identify the predominant S287-kinase as Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates Cdc25C efficiently on S287 in vitro and, like Chk1, is inhibited by 7-hydroxystaurosporine (UCN-01) and debromohymenialdisine, compounds that abrogate G2 arrest in somatic cells. CaMKII delays Cdc2/cyclin B activation via phosphorylation of Cdc25C at S287 in egg extracts, indicating that this pathway regulates the timing of mitosis during the early embryonic cell cycle.

Genomic Database Searching.

The availability of reference genome sequences for virtually all species under active research has revolutionized biology. Analyses of genomic variations in many organisms have provided insights into phenotypic traits, evolution and disease, and are transforming medicine. All genomic data from publicly funded projects are freely available in Internet-based databases, for download or searching via genome browsers such as Ensembl, Vega, NCBI's Map Viewer, and the UCSC Genome Browser. These online tools generate interactive graphical outputs of relevant chromosomal regions, showing genes, transcripts, and other genomic landmarks, and epigenetic features mapped by projects such as ENCODE.This chapter provides a broad overview of the major genomic databases and browsers, and describes various approaches and the latest resources for searching them. Methods are provided for identifying genomic locus and sequence information using gene names or codes, identifiers for DNA and RNA molecules and proteins; also from karyotype bands, chromosomal coordinates, sequences, motifs, and matrix-based patterns. Approaches are also described for batch retrieval of genomic information, performing more complex queries, and analyzing larger sets of experimental data, for example from next-generation sequencing projects.

Proteomic data on the nuclear interactome of human MCM9.

We present data relating to the interactome of MCM9 from the nuclei of human cells. MCM9 belongs to the AAA+ superfamily, and contains an MCM domain and motifs that may confer DNA helicase activity. MCM9 has been shown to bind MCM8, and has been implicated in DNA replication and homologous recombination. However, the mechanistic basis of MCM9's role in DNA repair is poorly understood, and proteins with which it interacts were hitherto unknown. We performed tandem affinity purification of MCM9 and its interacting proteins from nuclear extracts of human cells, followed by proteomic analysis, thereby generating a set of mass spectrometry data corresponding to the MCM9 interactome [1]. The proteomic data set comprises 29 mass spectrometry RAW files, deposited to the ProteomeXchange Consortium, and freely available from the PRIDE partner repository with the data set identifier PXD000212. A set of 22 interacting proteins identified from the proteomic data was used to create an MCM9-centered interactive network diagram, using the Cytoscape program. These data allow the scientific community to access, mine and explore the human nuclear MCM9 interactome.

Many fingers on the mitotic trigger: post-translational regulation of the Cdc25C phosphatase.

The mitotic inducer Cdc25 phosphatase controls the activation of Cdc2/cyclin B protein kinase and entry into mitosis in eukaryotic cells. Cdc25C is highly regulated by multiple post-translational modifications within its N-terminal regulatory domain and site-specific protein interactions. Phosphorylation of one inhibitory site targeted by multiple kinases determines the timing of Cdc25C activation and arrests cells in G2 in response to checkpoint, stress, developmental and extracellular signals. In mitosis, phosphorylation of several Ser/Thr residues and Pin1-catalysed peptidyl-proline isomerisation produces activation. Phosphorylation of one activating site is antagonistic to the proximal inhibitory site and maintains Cdc25C activity during mitosis. Phosphorylation and interacting proteins also modulate the nuclear import and export signals on Cdc25C, inducing dramatic changes in its localisation within the cell. Thus, the regulation of Cdc25C activity and localization integrates multiple signals that govern the decision to enter mitosis.

Dephosphorylation of the inhibitory phosphorylation site S287 in Xenopus Cdc25C by protein phosphatase-2A is inhibited by 14-3-3 binding.

Cdc25C phosphatase induces mitosis by dephosphorylating and activating Cdc2/cyclin B protein kinase. Phosphorylation of Xenopus Cdc25C at serine 287 creates a binding site for a 14-3-3 protein and restrains activation during interphase. Here, we show that dephosphorylation of S287 is catalysed by protein phosphatase-2A in Xenopus egg extracts. 14-3-3 protein binding to Cdc25C inhibits dephosphorylation of S287, providing a mechanism to maintain phosphorylation of that site during interphase. The rate of dephosphorylation of S287 is not increased in mitotic extracts, indicating that the phosphorylation status of the site is likely to be controlled through modulation of kinases or 14-3-3 binding activity.

What's that gene (or protein)? Online resources for exploring functions of genes, transcripts, and proteins.

The genomic era has enabled research projects that use approaches including genome-scale screens, microarray analysis, next-generation sequencing, and mass spectrometry-based proteomics to discover genes and proteins involved in biological processes. Such methods generate data sets of gene, transcript, or protein hits that researchers wish to explore to understand their properties and functions and thus their possible roles in biological systems of interest. Recent years have seen a profusion of Internet-based resources to aid this process. This review takes the viewpoint of the curious biologist wishing to explore the properties of protein-coding genes and their products, identified using genome-based technologies. Ten key questions are asked about each hit, addressing functions, phenotypes, expression, evolutionary conservation, disease association, protein structure, interactors, posttranslational modifications, and inhibitors. Answers are provided by presenting the latest publicly available resources, together with methods for hit-specific and data set-wide information retrieval, suited to any genome-based analytical technique and experimental species. The utility of these resources is demonstrated for 20 factors regulating cell proliferation. Results obtained using some of these are discussed in more depth using the p53 tumor suppressor as an example. This flexible and universally applicable approach for characterizing experimental hits helps researchers to maximize the potential of their projects for biological discovery.

Expression of the 49 human ATP binding cassette (ABC) genes in pluripotent embryonic stem cells and in early- and late-stage multipotent mesenchymal stem cells: possible role of ABC plasma membrane transporters in maintaining human stem cell pluripotency.

The 49-member human ATP binding cassette (ABC) gene family encodes 44 membrane transporters for lipids, ions, peptides or xenobiotics, four translation factors without transport activity, as they lack transmembrane domains, and one pseudogene. To understand the roles of ABC genes in pluripotency and multipotency, we performed a sensitive qRT-PCR analysis of their expression in embryonic stem cells (hESCs), bone marrow-derived mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We confirm that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs. We observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These variations are mainly due to plasma membrane transporters with low but significant gene expression: 18 are expressed in hESCs compared with 16 in hES-MSCs and 8 in hMSCs, suggesting important roles in pluripotency. Several of these ABCs shared similar substrates but differ regarding gene regulation. ABCA13 and ABCB4, similarly to ABCB1, could be new markers to select primitive hMSCs with specific plasma membrane transporter (low) phenotypes. ABC proteins performing basal intracellular functions, including translation factors and mitochondrial heme transporters, showed the highest constant gene expression among the three populations. Peptide transporters in the endoplasmic reticulum, Golgi and lysosome were well expressed in hESCs and slightly upregulated in hMSCs, which play important roles during the development of stem cell niches in bone marrow or meningeal tissue. These results will be useful to study specific cell cycle regulation of pluripotent stem cells or ABC dysregulation in complex pathologies, such as cancers or neurological disorders.

Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling.

The timing and localization of events during mitosis are controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2 (never in mitosis kinase 2), Plk1 (Polo-like kinase 1), and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by positional scanning oriented peptide library screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.

Systematic phosphorylation analysis of human mitotic protein complexes.

Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as "switchboards" to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1.

A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes.

The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004; Larsen et al., 2005; Kweon and Hakansson, 2006; Cantin et al., 2007; Collins et al., 2007). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO(2)-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO(2) and ZrO(2) columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.

Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells.

When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A-B55alpha complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A-B55alpha functions downstream of Cdk1 inactivation. PP2A-B55alpha isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-beta1, and RNAi depletion of importin-beta1 delayed mitotic exit synergistically with PP2A-B55alpha. This demonstrates that PP2A-B55alpha and importin-beta1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.