ABSTRACT
For initiation of eukaryotic DNA replication the origin recognition complex (ORC) associates with chromatin sites and constitutes a landing pad allowing Cdc6, Cdt1 and MCM proteins to accomplish the pre-replication complex (pre-RC). In S phase, the putative MCM helicase is assumed to move away from the ORC to trigger DNA unwinding. By using the fluorescence-based assays bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) we show in live mammalian cells that one key interaction in pre-RC assembly, the interaction between Orc2 and Orc3, is not restricted to the nucleus but also occurs in the cytoplasm. BRET assays also revealed a direct interaction between Orc2 and nuclear localization signal (NLS)-depleted Orc3. Further, we assessed the subcellular distribution of Orc2 and Orc3 in relation to MCM proteins Mcm3 and Mcm6 as well as to a key protein involved in elongation of DNA replication, proliferating nuclear cell antigen (PCNA). Our findings illustrate the spatial complexity of the elaborated process of DNA replication as well as that the BRET and BiFC techniques are novel tools that could contribute to our understanding of the processes at the very beginning of the duplication of the genome.
Subject(s)
DNA Replication , Origin Recognition Complex/metabolism , Animals , Biotechnology/methods , Cell Nucleus/metabolism , Cells, Cultured , Cytoplasm/metabolism , Eukaryotic Cells , Humans , Mice , Peptide Chain Elongation, Translational , Replication Origin , Spectrometry, Fluorescence/methods , TransfectionABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , Centromere/metabolism , DNA Replication/physiology , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Origin Recognition Complex/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Line , Cell Proliferation , Centromere/genetics , DNA Replication/genetics , DNA-Binding Proteins/genetics , Fluorescent Antibody Technique , Green Fluorescent Proteins , Mice , Minichromosome Maintenance Complex Component 3 , Mitosis/genetics , Mitosis/physiology , Nuclear Proteins/genetics , Origin Recognition Complex/genetics , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Tubulin/genetics , Tubulin/metabolism , Polo-Like Kinase 1ABSTRACT
OBJECTIVES: To determine the degree of association of five single nucleotide polymorphisms at the 5'-untranslated region (5'-UTR) of the human dopamine transporter gene (hSLC6A3; hDAT1) in bipolar affective disorder. METHODS: In a case-control design study, the polymorphisms were genotyped for allelic and genotypic distribution between 105 index cases (50 males) with bipolar affective disorder according to DSM IV and 199 unaffected control subjects (120 males). RESULTS: At the 5'-UTR locus of hSLC6A3, no significant allelic or genotypic differences were observed between index cases and controls. However, distinct 5-locus genotypes accumulated in subjects with bipolar affective disorder compared to control subjects (p = 0.029, odds ratio 1.84, 95% confidence interval 1.12-3.02). CONCLUSIONS: In conclusion, our data do not provide evidence for a major role of the 5'-UTR of the dopamine transporter gene in bipolar affective disorder. A minor contribution of distinct genotypes may be possible and warrants replication in extended samples.
Subject(s)
5' Untranslated Regions/genetics , Bipolar Disorder/genetics , Dopamine Plasma Membrane Transport Proteins/genetics , Polymorphism, Restriction Fragment Length , Adult , Alleles , Bipolar Disorder/diagnosis , Bipolar Disorder/psychology , Case-Control Studies , Chromosome Mapping , Female , Genotype , Humans , Male , Middle Aged , Promoter Regions, GeneticABSTRACT
BACKGROUND: The chromosome 22q11 region is proposed as a major candidate locus for susceptibility genes to schizophrenia. Recently, the gene ZDHHC8 encoding a putative palmitoyltransferase at 22q11 was proposed to increase liability to schizophrenia based on both animal models and human association studies by significant over-transmission of allele rs175174A in female, but not male subjects with schizophrenia. METHODS: Given the genetic complexity of schizophrenia and the potential genetic heterogeneity in different populations, we examined rs175174 in 204 German proband-parent triads and in an independent case-control study (schizophrenic cases: n = 433; controls: n = 186). RESULTS: In the triads heterozygous parents transmitted allele G preferentially to females, and allele A to males (heterogeneity chi2 = 4.43; p = 0.035). The case-control sample provided no further evidence for overall or gender-specific effects regarding allele and genotype frequency distributions. CONCLUSION: The findings on rs175174 at ZDHHC8 are still far from being conclusive, but evidence for sexual dimorphism is moderate, and our data do not support a significant genetic contribution of rs175174 to the aetiopathogenesis of schizophrenia.
