The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association.
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Saccharomyces cerevisiae Cdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition. (+info)
The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks.
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The origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae as a protein that specifically binds to origins of DNA replication. Although ORC appears to play an essential role in the initiation of DNA replication in the cells of all eukaryotes, its interactions with DNA have not been defined in species other than budding yeast. We have characterized a Schizosaccharomyces pombe homologue of the ORC subunit, Orc4p. The homologue (Orp4p) consists of two distinct functional domains. The C-terminal domain shows strong sequence similarity to human, frog, and yeast Orc4 proteins, including conserved ATP-binding motifs. The N-terminal domain contains nine copies of the AT-hook motif found in a number of DNA-binding proteins, including the members of the HMG-I(Y) family of chromatin proteins. AT-hook motifs are known from biochemical and structural studies to mediate binding to the minor groove of AT-tracts in DNA. Orp4p is essential for viability of Sc. pombe and is expressed throughout the cell cycle. The Orp4 protein (and its isolated N-terminal domain) binds to the Sc. pombe replication origin, ars1. The DNA binding properties of Orp4p provide a plausible explanation for the characteristic features of Sc. pombe origins of replication, which differ significantly from those of Sa. cerevisiae. (+info)
The BAH (bromo-adjacent homology) domain: a link between DNA methylation, replication and transcriptional regulation.
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Using sensitive methods of sequence analysis including hydrophobic cluster analysis, we report here a hitherto undescribed family of modules, the BAH (bromo-adjacent homology) family, which includes proteins such as eukaryotic DNA (cytosine-5) methyltransferases, the origin recognition complex 1 (Orc1) proteins, as well as several proteins involved in transcriptional regulation. The BAH domain appears to act as a protein-protein interaction module specialized in gene silencing, as suggested for example by its interaction within yeast Orc1p with the silent information regulator Sir1p. The BAH module might therefore play an important role by linking DNA methylation, replication and transcriptional regulation. (+info)
ORC localization in Drosophila follicle cells and the effects of mutations in dE2F and dDP.
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We isolated mutations in Drosophila E2F and DP that affect chorion gene amplification and ORC2 localization in the follicle cells. In the follicle cells of the ovary, the ORC2 protein is localized throughout the follicle cell nuclei when they are undergoing polyploid genomic replication, and its levels appear constant in both S and G phases. In contrast, when genomic replication ceases and specific regions amplify, ORC2 is present solely at the amplifying loci. Mutations in the DNA-binding domains of dE2F or dDP reduce amplification, and in these mutants specific localization of ORC2 to amplification loci is lost. Interestingly, a dE2F mutant predicted to lack the carboxy-terminal transcriptional activation and RB-binding domain does not abolish ORC2 localization and shows premature chorion amplification. The effect of the mutations in the heterodimer subunits suggests that E2F controls not only the onset of S phase but also origin activity within S phase. (+info)
E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila.
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Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins. Replication is regulated by the recruitment of additional factors such as CDC6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, we show that the level of the ORC1 subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, we find that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila. (+info)
Limitations of silencing at native yeast telomeres.
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Silencing at native yeast telomeres, in which the subtelomeric elements are intact, is different from silencing at terminal truncations. The repression of URA3 inserted in different subtelomeric positions at several chromosome ends was investigated. Many ends exhibit very little silencing close to the telomere, while others exhibit substantial repression in limited domains. Silencing at native ends is discontinuous, with maximal repression found adjacent to the ARS consensus sequence in the subtelomeric core X element. The level of repression declines precipitously towards the centromere. Mutation of the ARS sequence or an adjacent Abf1p-binding site significantly reduces silencing. The subtelomeric Y' elements are resistant to silencing along their whole length, yet silencing can be re-established at the proximal X element. Deletion of PPR1, the transactivator of URA3, and SIR3 overexpression do not increase repression or extend spreading of silencing to the same extent as with terminally truncated ends. sir1Delta causes partial derepression at X-ACS, in contrast to the lack of effect seen at terminal truncations. orc2-1 and orc5-1 have no effect on natural silencing yet cause derepression at truncated ends. X-ACS silencing requires the proximity of the telomere and is dependent on SIR2, SIR3, SIR4 and HDF1. The structures found at native yeast telomeres appear to limit the potential of repressive chromatin. (+info)
Assembly of functionally active Drosophila origin recognition complex from recombinant proteins.
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In eukaryotes the sites for the initiation of chromosomal DNA replication are believed to be determined in part by the binding of a heteromeric origin recognition complex (ORC) to DNA. We have cloned the genes encoding the subunits of the Drosophila ORC. Each of the genes is unique and can be mapped to discrete chromosomal locations implying that the pattern and developmental regulation of origin usage in Drosophila is not regulated solely by a large family of different ORC proteins. The six-subunit ORC can be reconstituted with recombinant proteins into a complex that restores DNA replication in ORC-depleted Drosophila or Xenopus egg extracts. (+info)
A role for a replicator dominance mechanism in silencing.
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The role of the natural HMR-E silencer in modulating replication initiation and silencing by the origin recognition complex (ORC) was examined. When natural HMR-E was the only silencer controlling HMR, the silencer's ORC-binding site (ACS) was dispensable for replication initiation but essential for silencing, indicating that a non-silencer chromosomal replicator(s) existed in close proximity to the silencer. Further analysis revealed that regions flanking both sides of HMR-E contained replicators. In contrast to replication initiation by the intact silencer, initiation by the non-silencer replicator(s) was abolished in an orc2-1 mutant, indicating that these replicators were extremely sensitive to defects in ORC. Remarkably, the activity of one of the non-silencer replicators correlated with reduced silencing; inactivation of these replicators caused by either the orc2-1 mutation or the deletion of flanking sequences enhanced silencing. These data were consistent with a role for the ORC bound to the HMR-E silencer ACS in suppressing the function of neighboring ORC molecules capable of inhibiting silencing, and indicated that differences in ORC-binding sites within HMR itself had profound effects on ORC function. Moreover, replication initiation by natural HMR-E was inefficient, suggesting that closely spaced replicators within HMR contributed to an inhibition of replication initiation. (+info)