Cdc7 Mutants Research Articles

Modulation of Gene Silencing by Cdc7p via H4 K16 Acetylation and Phosphorylation of Chromatin Assembly Factor CAF-1 in Saccharomyces cerevisiae.

CAF-1 is an evolutionarily conserved H3/H4 histone chaperone that plays a key role in replication-coupled chromatin assembly and is targeted to the replication fork via interactions with PCNA, which, if disrupted, leads to epigenetic defects. In Saccharomyces cerevisiae, when the silent mating-type locus HMR contains point mutations within the E silencer, Sir protein association and silencing is lost. However, mutation of CDC7, encoding an S-phase-specific kinase, or subunits of the H4 K16-specific acetyltransferase complex SAS-I, restore silencing to this crippled HMR, HMRae** Here, we observed that loss of Cac1p, the largest subunit of CAF-1, also restores silencing at HMRae**, and silencing in both cac1Δ and cdc7 mutants is suppressed by overexpression of SAS2 We demonstrate Cdc7p and Cac1p interact in vivo in S phase, but not in G1, consistent with observed cell cycle-dependent phosphorylation of Cac1p, and hypoacetylation of chromatin at H4 K16 in both cdc7 and cac1Δ mutants. Moreover, silencing at HMRae** is restored in cells expressing cac1p mutants lacking Cdc7p phosphorylation sites. We also discovered that cac1Δ and cdc7-90 synthetically interact negatively in the presence of DNA damage, but that Cdc7p phosphorylation sites on Cac1p are not required for responses to DNA damage. Combined, our results support a model in which Cdc7p regulates replication-coupled histone modification via a CAC1-dependent mechanism involving H4 K16ac deposition, and thereby silencing, while CAF-1-dependent replication- and repair-coupled chromatin assembly per se are functional in the absence of phosphorylation of Cdc7p consensus sites on CAF-1.

Characterization of a Drosophila Ortholog of the Cdc7 Kinase: A ROLE FOR Cdc7 IN ENDOREPLICATION INDEPENDENT OF CHIFFON

Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-replication complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G1/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has yet been characterized. Here, we report the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification but still undergo endocycling. Our results show that Cdc7 interacts with Chiffon to form a functional Dbf4-dependent kinase complex and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, we show that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila.

Identification and Characterization of Human Cdc7 Nuclear Retention and Export Sequences in the Context of Chromatin Binding

The Cdc7 serine/threonine kinase activates the initiation of DNA replication by phosphorylating MCM proteins that are bound to the origins of DNA replication. We reported previously that human Cdc7 nuclear import is mediated directly by importin-beta through its binding to the Cdc7 nuclear localization sequence (NLS). Here, we report that human Cdc7 nuclear localization is regulated by two additional elements: nuclear retention (NRS) and export sequences (NES). Cdc7 proteins imported into the nucleus are retained in the nucleus by associating with chromatin, for which NRS-(306-326) is essential. Importantly, this binding appears to be specific to the origin of DNA replication, because the binding of wild-type Cdc7 to origin is 2.4-fold higher than to non-origin DNA. Furthermore, an NRS-defective Cdc7 mutant could not be retained in the nucleus, although it was imported into the nucleus normally. Together, our data suggest that NRS plays an important role in the activation of DNA replication by Cdc7. The Cdc7 proteins unassociated with chromatin are bound by CRM1 via two NES elements: NES1 at 458-467 within kinase insert III, and NES2 at 545-554 within the kinase IX domain. The primary function of the Cdc7-CRM1 association may be to translocate nuclear Cdc7 to the cytoplasm. However, the binding of CRM1 with Cdc7 at NES2 raises an interesting possibility that CRM1 may also down-regulate Cdc7 by masking its kinase domain.

Human and Xenopus cDNAs encoding budding yeast Cdc7-related kinases: in vitro phosphorylation of MCM subunits by a putative human homologue of Cdc7.

Saccharomyces cerevisiae Cdc7 kinase is essential for initiation of DNA replication, and Hsk1, a related kinase of Schizosaccharomyces pombe, is also required for DNA replication of fission yeast cells. We report here cDNAs encoding Cdc7-related kinases from human and Xenopus (huCdc7 and xeCdc7, respectively). The cloned cDNA for huCdc7 contains an open reading frame consisting of 574 amino acids with a predicted molecular weight of 63,847 that possesses overall amino acid identity of 32% (54% including similar residues) to Cdc7 and Hsk1. huCDC7 is transcribed in the various tissues examined, but most abundantly in testis. Three transcripts of 4.4, 3.5 and 2.4 kb in length are detected. The 3.5 kb transcript is the most predominant and is expressed in all the tissues examined. A cDNA containing a 91 nucleotide insertion at the N-terminal region of huCDC7 is also detected, suggesting the presence of multiple splicing variants. The huCdc7 protein is expressed at a constant level during the mitotic cell cycle and is localized primarily in nuclei in interphase and distributed diffusibly in cytoplasm in the mitotic phase. The wild-type huCdc7 protein expressed in COS7 cells phosphorylates MCM2 and MCM3 proteins in vitro, suggesting that huCdc7 may regulate processes of DNA replication by modulating MCM functions.

Analyses of Saccharomyces cerevisiae Cdc7 kinase point mutants: dominant-negative inhibition of DNA replication on overexpression of kinase-negative Cdc7 proteins.

Saccharomyces cerevisiae Cdc7 kinase is required for initiation of S phase, and its kinase activity, which is positively regulated by Dbf4 protein, reaches maximum at the G1/S boundary. In this study, we constructed Cdc7 point mutants (T281E, T281A, D182N, D163N, and T167E) and examined the effect of each mutant on growth. All the mutants lost the ability to complement temperature-sensitive growth of cdc7(ts) mutants at a low protein level, whereas T281A (putative target of phosphorylation) and T167E (residue involved in substrate recognition) restored the growth of cdc7(ts) when overproduced to a high level. Three putative kinase-negative mutants (T281E, D182N, and D163N) inhibited growth when overexpressed in a wild-type strain. Analyses of DNA content and morphology revealed that most cells were arrested as dumbbells with 1C DNA, indicative of a block in the G1 to S transition. This growth inhibition was suppressed by co-overexpression of the wild-type Cdc7 or Dbf4 protein. Furthermore, deletion of the Dbf4 protein-binding region in each Cdc7 mutant resulted in loss of growth inhibitory effect. Thus, dominant-negative effects of T281E, D182N, and D163N on growth can be best explained by inactivation of the wild-type Cdc7 function through titration of Dbf4 by these inactive kinases. Our results are consistent with the notion that association of Dbf4 with Cdc7 is essential for the G1 to S transition in S. cerevisiae.

Preparation of active Cdc7/Dbf4 kinase from yeast cells

This chapter discusses the preparation of active cdc7/Dbf4 kinase from yeast cells. Saccharomyces cerevisiae cdc7 mutants are blocked in the cell cycle at the G1/S boundary at the restrictive temperature. Biochemical studies have shown that Cdc7 is a serine/threonine protein kinase that phosphorylates histone H1. It has been proposed that Cdc7 kinase is a component, along with Clb5,6/Cdc28 kinase, of S phase-promoting factor (SPF). Thus, there is great interest in determining what activates Cdc7 kinase at G1/S 5 and in defining its in vivo substrates. Progress in characterizing the kinase in these respects has been limited, however, not only because it is active only transiently during the cell cycle and is present only at low levels in the cell, but also because it has been unexpectedly difficult to express active Cdc7 in available expression systems and because the only test substrate, histone H1, is not an efficient substrate.

Genetic interactions between CDC7 and CDC28: growth inhibition of cdc28-1N by Cdc7 point mutants.

Cdc7 kinase of Saccharomyces cerevisiae, a nuclear phosphoprotein, regulates initiation of chromosomal DNA replication. Overexpression of kinase-negative Cdc7 point mutants (T281E, D182N and D163N) arrests the cell cycle of the wild-type Saccharomyces cerevisiae cells at the G1/S boundary. This is caused by titration of a regulatory protein, Dbf4, from the wild-type Cdc7, which leads to inactivation of its kinase activity. We report here that kinase-negative Cdc7 mutants, when overexpressed in cdc28-1N(ts) at a permissive temperature, not only inhibit DNA replication by inactivating the wild-type Cdc7 but may also disturb coordination between DNA replication and cell division. Suppression of growth inhibition under this condition requires co-expression of both Dbf4 and Cdc28, whereas Dbf4 alone can counteract the growth inhibition in the wild-type cells. In cdc28-1N(ts), co-expression of the wild-type Dbf4 rescues only the G1/S defect and results in accumulation of those cells with less than 1C DNA as well as 2C DNA. On the other hand, co-expression of Cdc28 alone leads to increase of those cells arrested at the G1/S boundary, as found typically in the wild-type. We also report that overexpression of T281A, a 'weak' allele of Cdc7, causes growth arrest in cdc28-1N(ts) cells, but not in the CDC28 wild-type cells. This suggests that T281A is inactive in cdc28-1N(ts) and is consistent with the idea that Cdc28 activates Cdc7 by phosphorylation. We conclude that two essential serine-threonine kinases, Cdc28 and Cdc7, genetically interact for initiation of the S phase and possibly for G2/M progression and/or S phase checkpoint control.

Pulsed Field Gel Electrophoresis Labeling Method to Study the Pattern of Saccharomyces cerevisiae Chromosomal DNA Synthesis During the G1/S Phase of the Cell Cycle

Yeast Saccharomyces cerevisiae is an excellent model to study eukaryotic DNA replication. Since yeast lacks thymidine kinase, it is difficult to assay DNA synthesis. In this report, a novel approach called the pulsed field gel electrophoresis (PFGE) labeling method is used to investigate yeast chromosomal DNA synthesis. In this method, yeast cells are first labeled by 32P in vivo and chromosomal DNA molecules are then resolved by pulsed field gel electrophoresis. A linear 32P labeling of chromosomal-size DNA molecules can be observed up to 100 min in an asynchronized culture. In an α-factor arresting-and-releasing synchronized culture, we observed that 32P can be rapidly taken up in the S phase. Our results show that all of the chromosomes are labeled at approximately the same time (within a 15-min interval), suggesting that the temporal order of all chromosomal DNA synthesis is synchronized in the S phase. Cell cycle blockers were used to arrest the yeast cultures to study DNA synthesis that coincides with cell cycle analysis. Our results show that α-factor and hydroxyurea block the cell cycle at the late G1 and S phases, respectively, resulting in the failure of DNA synthesis. Nocodazole blocks the cell cycle after the S phase and thus shows no effect on chromosomal DNA synthesis. Since the PFGE labeling method is highly specific to chromosomal DNA synthesis, we used it to examine the DNA synthesis pattern in various cell cycle mutants. All of the G1/S cdc mutants tested, cdc4, 6, 7, 8, 17, 2, and 9, cannot label the chromosomal size of DNA molecules; but the G2/M cdc mutant cdc13 can. Specific findings are (i) cdc9 and cdc7 mutant cells do not synthesize chromosomal DNA molecules, although some 32P incorporation could be observed at nonpermissive temperature; (ii) cdc6 is confirmed to be defective in DNA synthesis; and (iii) all three yeast polymerases are defective in DNA synthesis, suggesting their role(s) are in the elongation step. Thus, the use of the PFGE labeling method combined with genetic studies in yeast would facilitate exploration of the mechanism(s) of DNA replication in eukaryotes.

Yeast pre-meiotic DNA replication utilizes mitotic origin ARS1 independently of CDC7 function

In budding yeast, mitotic DNA replication initiates at sequence-specific replication origins, the prototype for which is ARS1. Initiation serves as the primary control point for mitotic DNA replication, and is catalyzed by the Cdc7 protein kinase. In contrast, premeiotic DNA replication apparently does not require Cdc7, and the existence and nature of specific replication origins in the meiotic division cycle have not been previously reported. We have begun to investigate the mechanism of premeiotic DNA synthesis by determining whether or not ARS1 functions as a DNA replication origin in meiosis. We have taken advantage of the fact that transcription through ARS1 disrupts its ability to function as an origin to show that ARS1 is required for premeiotic DNA replication of a plasmid bearing this element. Further, premeiotic replication from ARS1 still occurs in a cdc7 mutant strain held at conditions non-permissive for Cdc7 protein kinase activity. These findings reveal that premeiotic DNA replication can initiate from origins also used in mitosis, and is not regulated by Cdc7. Taken together with previous findings implicating Cdc7 in meiotic DNA recombination and induced mutagenesis, these findings prompt us to postulate that the Cdc7 protein kinase regulates some step common to several DNA metabolic processes such as local disassembly of chromatin or activation of a key component of the DNA metabolic machinery.

Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast.

The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.

DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase.

The yeast Cdc7 protein is indispensable to initiation of nuclear DNA replication, based on the phenotype of the conditional, temperature-sensitive (ts) cdc7 mutants at the restrictive temperature. This protein has likewise been implicated in commitment to meiotic DNA recombination and induced mutagenesis, which may result from error-prone DNA repair. Our previous work revealed sequence similarity between the Cdc7 protein and known protein kinases. To determine whether it possesses kinase activity, we have immunoprecipitated the protein from Cdc7-overproducing yeast cells by using polyclonal antibodies raised against a nondenatured beta-galactosidase-Cdc7 fusion protein. In this report, we demonstrate that Cdc7 immune complexes are capable of phosphorylating mammalian histone H1 on serine and/or threonine residues. Immune complexes derived from cells harboring the cdc7-2 ts mutant gene on a high copy number plasmid possess a thermolabile kinase activity. Thus, we postulate that Cdc7 may regulate the various DNA metabolic pathways by phosphorylating one or more target substrates. Because Cdc7 kinase acts downstream of Cdc28/cdc2 kinase function at "start," the transition from G1 to S phase in the cell cycle may be the result of a cascade of protein phosphorylation.

The possible functional significance of phosphatidylinositol in G 1 arrest of Saccharomyces cerevisiae

Individual phospholipids were assayed in exponentially growing and G 1-arrested temperature-sensitive cell division cycle ( cdc) mutants of Saccharomyces cerevisiae. It was observed that cdc28 cells which are known to arrest at ‘start’ when shifted to their non-permissive temperature, resulted in a 40% decrease in phosphatidylinositol (PI) level while the phosphatidylserine (PS) content was doubled in these cells. The reduced level of PI was restored in cdc4 and cdc7 mutants which are known to arrest past the ‘start’. The increase in PS level in cdc8 mutant which was probably to compensate the intrinsic charging of membrane environment, was also reduced in cdc4 and cdc7 mutants. Our results demonstrate that PI may play a role in yeast cell division and growth that the abnormalities of cdc28 could also be related to PI decrease.