The human and murine MOK2 proteins are factors able to recognize both DNA and RNA through their zinc finger motifs. This dual affinity of MOK2 suggests that MOK2 might be involved in transcription and post-transcriptional regulation of MOK2 target genes. The IRBP gene contains two MOK2-binding elements, a complete 18 bp MOK2-binding site located in intron 2 and the essential core MOK2-binding site (8 bp of conserved 3'-half-site) located in the IRBP promoter. We have demonstrated that MOK2 can bind to the 8 bp present in the IRBP promoter and repress transcription from this promoter by competing with the CRX activator for DNA binding. In this study, we identify a novel interaction between lamin A/C and hsMOK2 by using the yeast two-hybrid system. The interaction, which was confirmed by GST pull-down assays and co-immunolocalization studies in vivo, requires the N-terminal acidic domain of hsMOK2 and the coiled 2 domain of lamin A/C. Furthermore, we show that a fraction of hsMOK2 protein is associated with the nuclear matrix. We therefore suggest that hsMOK2 interactions with lamin A/C and the nuclear matrix may be important for its ability to repress transcription.
The POL1 gene of the fission yeast, Schizosaccharomyces pombe, was isolated using a POL1 gene probe from the budding yeast Saccharomyces cerevisiae, cloned and sequenced. This gene is unique and located on chromosome II. It includes a single 91 bp intron and is transcribed into a mRNA of about 4500 nucleotides. The predicted protein coded for by the S. pombe POL1 gene is 1405 amino acid long and its calculated molecular weight is about 160,000 daltons. This peptide contains seven amino acid blocks conserved among several DNA polymerases from different organisms and shares overall 37% and 34% identity with DNA polymerases alpha from S. cerevisiae and human cells, respectively. These results indicate that this gene codes for the S. pombe catalytic subunit of DNA polymerase alpha. The comparisons with human DNA polymerase alpha and with the budding yeast DNA polymerases alpha, delta and epsilon reveal conserved blocks of amino acids which are structurally and/or functionally specific only for eukaryotic alpha-type DNA polymerases.
Background information. hsMOK2 (human MOK2) is a DNA-binding transcriptional repressor. For example, it represses the IRBP (interphotoreceptor retinoid-binding protein) gene by competing with the CRX (cone-rod homeobox protein) transcriptional activator for DNA binding. Previous studies have shown an interaction between hsMOK2 and nuclear lamin A/C. This interaction could be important to explain hsMOK2 ability to repress transcription.Results. In the present study, we have tested whether missense pathogenic mutations of lamin A/C, which are located in the hsMOK2-binding domain, could affect the interaction with hsMOK2. We find that none of the tested mutations is able to disrupt hsMOK2 binding in vitro or in vivo. However, we observe an aberrant cellular localization of hsMOK2 into nuclear aggregates when pathogenic lamin A/C mutant proteins are expressed.Conclusions. These results indicate that pathogenic mutations in lamin A/C lead to sequestration of hsMOK2 into nuclear aggregates, which may deregulate MOK2 target genes.
We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase delta. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2+/rhp9+. This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30 degrees C, but were defective in this checkpoint function when treated with MMS at 37 degrees C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37 degrees C. Furthermore, the crb2+ gene was required, together with the cds1+ gene, for the S/M checkpoint at 30 degrees C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the poldeltats3 background at both 30 degrees C and 37 degrees C. The rapid death phenotype was independent of the checkpoint functions.
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