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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The human and murine MOK2 orthologue genes encode Krü ppel/TFIIIA-related zinc finger proteins, which are factors able to recognize both DNA and RNA through their zinc finger motifs. MOK2 proteins have been shown to bind to the same 18-base pair (bp)-specific sequence in duplex DNA. This MOK2-binding site was found within introns 7 and 2 of human PAX3 and interphotoreceptor retinoid-binding protein (IRBP) genes, respectively. As these two genes are expressed in the brain as MOK2, we have suggested that PAX3 and IRBP genes are two potentially important target genes for the MOK2 protein. In this study, we focused our attention on IRBP as a potential MOK2 target gene. Sequence comparison and binding studies of the 18-bp MOK2-binding sites present in intron 2 of human, bovine, and mouse IRBP genes show that the 3-half sequence is the essential core element for MOK2 binding. Very interestingly, 8-bp of this core sequence are found in a reverse orientation, in the IRBP promoter. We demonstrate that MOK2 can bind to the 8-bp sequence present in the IRBP promoter and repress its transcription when transiently overexpressed in retinoblastoma Weri-RB1 cells. In the IRBP promoter, it appears that the TAAAGGCT MOK2-binding site overlaps with the photoreceptor-specific CRX-binding element. We suggest that MOK2 represses transcription by competing with the cone-rod homeobox protein (CRX) for DNA binding, thereby decreasing transcriptional activation by CRX. Furthermore, we show that Mok2 expression in the developing mouse and in the adult retina seems to be concordant with IRBP expression.The human and murine MOK2 orthologue genes, which are preferentially expressed in brain and testis tissues, encode two different Krü ppel/TFIIIA-related zinc finger proteins. The human and murine genes have been localized to band q13.2-q13.3 of chromosome 19 and chromosome 6, respectively (1, 2). The human hsMOK2 protein shows substantial differences with the murine MOK2 protein. The mouse MOK2 protein contains seven tandem zinc finger motifs with only five additional amino acids at its COOH-terminal end (3). The seven fingers motifs are highly similar to one another but are distinct from those of other zinc finger proteins. The structural feature of murine MOK2 protein is also found at the end of human hsMOK2 protein. Furthermore, the human protein contains three additional zinc finger motifs in tandem with the others and a nonfinger acidic domain of 173 amino acids at the NH 2 -terminal end (2). We have previously shown that human MOK2 RNA maturation results in three mRNAs with different 5Ј-untranslated exons. One of these three mRNAs encodes a smaller MOK2 protein (hsMOK2⌬) containing 10 zinc finger motifs and a small NH 2 -acidic domain made up of 76 amino acids. We have shown that the human and murine MOK2 proteins are able to recognize both DNA and RNA through their zinc finger motifs (4). Electron microscopy and specific RNA homopolymer binding activity showed clearly that the murine and human MOK2 proteins are RNA-binding proteins th...

Background: Chk1 kinase is activated by phosphorylation at serine-345 by Rad3 checkpoint kinase and is required for DNA damage checkpoint in late S and G2 phase of S. pombe cell cycle. We studied the ability of two chk1 mutants, chk1-1 and chk1-2, to undergo phosphorylation and to delay cell cycle progression in response to different types of DNA lesions.

The zinc-finger transcription factor MOK2 recognizes both DNA and RNA through its zinc-finger motifs [1]. This dual affinity suggests that MOK2 may play a role in transcription, as well as in the post-transcriptional regulation of specific genes. We have shown that MOK2 represses expression of the interphotoreceptor retinoid-binding protein (IRBP) gene [2]. IRBP contains two MOK2-binding elements, a complete 18-bp MOK2-binding site, located in intron 2, and the essential 8-bp core MOK2-binding site (corresponding to the conserved 3¢-half site) which is in the IRBP promoter. MOK2 can bind to the 8-bp sequence in the IRBP promoter and repress transcription from this promoter. In the IRBP promoter, the TAAAGGCT MOK2-binding site overlaps with the photoreceptorspecific Crx-binding element, suggesting that MOK2 represses transcription by competing with the cone-rod homeobox protein for DNA binding and decreasing transcriptional activation by the cone-rod homeobox protein. The particular arrangement of the two MOK2-binding sites, observed in the human IRBP gene and also in a second human potential MOK2 target gene, Pax3, suggests that MOK2 may repress transcription via a dual mechanism. Previously, we identified lamin A ⁄ C proteins as binding partners for hsMOK2 in a yeast two-hybrid screen [3]. A-type lamins have been shown to bind hsMOK2 in vitro and in vivo through the coil 2 domain common to lamin A and lamin C, whereas the lamin A ⁄ C-binding site in hsMOK2 has been mapped to its N-terminal acidic domain. Divergent evolution has been observed between human and mouse MOK2 genes which results in the loss of this NH 2 -domain in the mouse gene [4]. An in silico search of MOK2 genes in different species has shown that the lamin-binding site is present only in primate MOK2 proteins. Furthermore, we have found that a fraction of human hsMOK2 protein is associated with the nuclear matrix. We therefore suggested that hsMOK2 interactions with lamin A ⁄ C and the nuclear matrix might be important for its