Biomedical subjects

Friedrich Grummt

Publications and source records attributed to Friedrich Grummt.

5 recordsLinked to original sources

PubMed2006

Mouse pre-replicative complex proteins colocalise and interact with the centrosome.

Initiation of eukaryotic DNA replication is achieved by the sequential binding of different proteins to origins of DNA replication. Using EGFP-tagged initiator proteins and immunofluorescence techniques we found that most of the ORC and the MCM subunits are localised at centrosomes and are colocalised with the polo-like protein kinase, Plk1. Yeast two-hybrid studies revealed interactions of Plk1 with the Mcm2 as well as the Orc2 protein. Co-immunoprecipitations showed an interaction of Plk1 with Mcm2 as well as interactions of gamma-tubulin with Mcm3 and Orc2, respectively. An in vitro phosphorylation assay showed that the Orc2 protein is a substrate of Plk1. Depletion of Orc2 and Mcm3 by siRNA leads to an inhibition of cell proliferation, an altered cell cycle distribution as well as to multinucleated cells with insufficiently organised microtubules. These results indicate an important role of the MCM and ORC proteins in mitosis besides their described role in the establishment of the pre-replicative complex.

Animals↗

PubMed2006

Interaction between HP1alpha and replication proteins in mammalian cells.

HP1 is an essential heterochromatin-associated protein known to play an important role in the organization of heterochromatin as well as in the transcriptional regulation of heterochromatic and euchromatic genes both in repression and activation. Using the yeast two-hybrid system and immunoprecipitation, we report here that murine HP1alpha interacts with the preRC proteins ORC1, ORC2 and CDC6. Immunofluorescence staining and EGFP/DsRed fusion proteins revealed a colocalization of HP1alpha with ORC1, ORC2 and CDC6 in heterochromatin, supporting the notion that ORC and probably CDC6 play an important role in murine HP1alpha function. Besides that, we also observed a colocalization of HP1alpha with gamma-tubulin suggesting a centrosomal localization of HP1alpha in murine cells. To gain insight into HP1alpha function, we applied the RNAi technique. Depletion of HP1alpha leads to a slow down of cell proliferation, an aberrant cell cycle progression as well as to multinucleated cells with insufficiently organized microtubule. These results together indicate that HP1alpha exerts functions in mitosis and cytokinesis.

Animals↗

PubMed2003

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells.

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.

Animals↗

PubMed2002

Transcription termination factor TTF-I exhibits contrahelicase activity during DNA replication.

In mammals, sequence-specific termination of DNA replication within the ribosomal RNA genes is catalyzed by a defined DNA-protein complex that includes transcription termination factor I (TTF-I). Here we show that TTF-I acts as a polar contrahelicase contrary to the intrinsic 3' -->5' helicase activity of SV40 large T antigen. The contrahelicase activity requires binding of TTF-I to its cognate recognition site and the presence of an auxiliary GC-rich sequence, which is able to form a specific secondary structure. Mutations in the GC-rich sequence lead to a loss of folding into correct secondary structure and abrogate contrahelicase activity. The finding suggests that a specific interaction between the Sal box-bound TTF-I and the GC-rich sequence is essential for the inhibition of T antigen helicase. Analyses of N-terminally truncated mutants of TTF-I showed inhibition of helicase by the same domain of TTF-I, which is also responsible for replication fork arrest.

Amino Acid Sequence↗

PubMed2002

Ku antigen supports termination of mammalian rDNA replication by transcription termination factor TTF-I.

A replication fork barrier at the 3'-end of mouse ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. This barrier is an inherent property of a defined DNA-protein complex including transcription termination factor I, and specific protein-protein interactions occur between this factor and protein(s) of the replication machinery. Here we report that a second DNA-binding protein is essential for barrier activity. We have purified and functionally characterised the protein from HeLa cells. The final preparation contained two polypeptides with molecular masses of 70 and 86 kDa, respectively. Both polypeptides interact with a GC-stretch adjacent to the binding site of transcription termination factor I. The specificity of binding to the barrier DNA was demonstrated in an electrophoretic mobility shift assay. The biochemical properties of this protein resemble that of Ku antigen, a human nuclear DNA-binding heterodimer that is the target of autoimmune-antibodies in several autoimmune diseases. Recombinant Ku protein, purified as heterodimer from co-infected insect cells, is able to partially rescue the barrier activity in Ku-depleted HeLa cell extracts. These data demonstrate that transcription termination factor I and Ku act synergistically to prevent head-on collision between the replication and the transcription machinery.

Animals↗