Biomedical subjects

S Urbach

Publications and source records attributed to S Urbach.

5 recordsLinked to original sources

PubMed2001

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.

Amino Acid Motifs↗

PubMed2001

The TRANSFAC system on gene expression regulation.

The TRANSFAC database on transcription factors and their DNA-binding sites and profiles (http://www.gene-regulation.de/) has been quantitatively extended and supplemented by a number of modules. These modules give information about pathologically relevant mutations in regulatory regions and transcription factor genes (PathoDB), scaffold/matrix attached regions (S/MARt DB), signal transduction (TRANSPATH) and gene expression sources (CYTOMER). Altogether, these distinct database modules constitute the TRANSFAC system. They are accompanied by a number of program routines for identifying potential transcription factor binding sites or for localizing individual components in the regulatory network of a cell.

Binding Sites↗

PubMed2000

Biochemical characterization of the Arabidopsis K+ channels KAT1 and AKT1 expressed or co-expressed in insect cells.

KAT1 and AKT1 belong to the multigenic family of the inwardly rectifying Shaker-like plant K+ channels. They were biochemically characterized after expression in insect cells using recombinant baculoviruses. The channels were solubilized from microsomal fractions prepared from infected cells (among eight different detergents only one, L-alpha-lysophosphatidylcholine, was efficient for solubilization), and purified to homogeneity using immunoaffinity (KAT1) or ion-exchange and size exclusion (AKT1) techniques. The following results were obtained with the purified polypeptides: (i) neither KAT1 nor AKT1 was found to be glycosylated; (ii) both polypeptides were mainly present as homotetrameric structures, supporting the hypothesis of a tetrameric structure for the functional channels; (iii) no heteromeric KAT1/AKT1 assembly was detected when the two polypeptides were co-expressed in insect cells. The use of the two-hybrid system in yeast also failed to detect any interaction between KAT1 and AKT1 polypeptides. Because of these negative results, the hypothesis that plant K+-channel subunits are able to co-assemble without any discrimination, previously put forward based on co-expression in Xenopus oocytes of various K+-channel subunits (including KAT1 and AKT1), has still to be supported by independent approaches. Co-localization of channel subunits within the same plant tissue/cell does not allow us to conclude that the subunits form heteromultimeric channels.

Animals↗

PubMed1997

Tetramerization of the AKT1 plant potassium channel involves its C-terminal cytoplasmic domain.

All plant channels identified so far show high conservation throughout the polypeptide sequence except in the ankyrin domain which is present only in those closely related to AKT1. In this study, the architecture of the AKT1 protein has been investigated. AKT1 polypeptides expressed in the baculovirus/Sf9 cells system were found to assemble into tetramers as observed with animal Shaker-like potassium channel subunits. The AKT1 C-terminal intracytoplasmic region (downstream from the transmembrane domain) alone formed tetrameric structures when expressed in Sf9 cells, revealing a tetramerization process different from that of Shaker channels. Tests of subfragments from this sequence in the two-hybrid system detected two kinds of interaction. The first, involving two identical segments (amino acids 371-516), would form a contact between subunits, probably via their putative cyclic nucleotide-binding domains. The second interaction was found between the last 81 amino acids of the protein and a region lying between the channel hydrophobic core and the putative cyclic nucleotide-binding domain. As the interacting regions are highly conserved in all known plant potassium channels, the structural organization of AKT1 is likely to extend to these channels. The significance of this model with respect to animal cyclic nucleotide-gated channels is also discussed.

Amino Acid Sequence↗

PubMed1996

The baculovirus/insect cell system as an alternative to Xenopus oocytes. First characterization of the AKT1 K+ channel from Arabidopsis thaliana.

Two plant (Arabidopsis thaliana) K+ transport systems, KAT1 and AKT1, have been expressed in insect cells (Sf9 cell line) using recombinant baculoviruses. Microscopic observation after immunogold staining revealed that the expressed AKT1 and KAT1 polypeptides were mainly associated with internal membranes, but that a minute fraction was targeted to the cell membrane. KAT1 was known, from earlier electrophysiological characterization in Xenopus oocytes, to be an inwardly rectifying voltage-gated channel highly selective for K+, while similar experiments had failed to characterize AKT1. Insect cells expressing KAT1 displayed an exogenous inwardly rectifying K+ conductance reminiscent of that described previously in Xenopus oocytes expressing KAT1. Under similar conditions, cells expressing AKT1 showed a disturbed cell membrane electrical stability that precluded electrophysiological analysis. Use of a baculovirus transfer vector designed so as to decrease the expression level allowed the first electrophysiological characterization of AKT1. The baculovirus system can thus be used as an alternative method when expression in Xenopus oocytes is unsuccessful for electrophysiological characterization of the ion channel of interest. The plant AKT1 protein has been shown in this way to be an inwardly rectifying voltage-gated channel highly selective for K+ ions and sensitive to cGMP.

Adenosine Triphosphate↗