Same partners, different dance: involvement of DNA replication proteins in centrosome regulation.

Genome replication is the most fundamental element of the continuity of life. In eukaryotes, DNA replication is regulated by an elegant network of many different protein factors to ensure the timely and accurate copying of their entire genome once per cell cycle. The replication factors include the maintenance (MCM) proteins, Cdt1, Cdc6, Cdc7, Cdc45, and geminin. All of these proteins are involved in the regulation of DNA replication at the initiation step. Interestingly, recent studies have shown that some of these replication proteins also localize to the centrosome, often throughout the entire cell cycle. These centrosomally localized replication proteins appear to play essential roles in the regulation of centrosome biogenesis, suggesting that genome replication and segregation are regulated interdependently. In this review, we summarize and discuss the inter-dependent regulation played by some of the replication proteins.

Interspecies variation reveals a conserved repressor of alpha-specific genes in Saccharomyces yeasts.

The mating-type determination circuit in Saccharomyces yeast serves as a classic paradigm for the genetic control of cell type in all eukaryotes. Using comparative genetics, we discovered a central and conserved, yet previously undetected, component of this genetic circuit: active repression of alpha-specific genes in a cells. Upon inactivation of the SUM1 gene in Saccharomyces bayanus, a close relative of Saccharomyces cerevisiae, a cells acquired mating characteristics of alpha cells and displayed autocrine activation of their mating response pathway. Sum1 protein bound to the promoters of alpha-specific genes, repressing their transcription. In contrast to the standard model, alpha1 was important but not required for alpha-specific gene activation and mating of alpha cells in the absence of Sum1. Neither Sum1 protein expression, nor its association with target promoters was mating-type-regulated. Thus, the alpha1/Mcm1 coactivators did not overcome repression by occluding Sum1 binding to DNA. Surprisingly, the mating-type regulatory function of Sum1 was conserved in S. cerevisiae. We suggest that a comprehensive understanding of some genetic pathways may be best attained through the expanded phenotypic space provided by study of those pathways in multiple related organisms.

GINS, a central nexus in the archaeal DNA replication fork.

In eukaryotes, the GINS complex is essential for DNA replication and has been implicated as having a role at the replication fork. This complex consists of four paralogous GINS subunits, Psf1, Psf2, Psf3 and Sld5. Here, we identify an archaeal GINS homologue as a direct interaction partner of the MCM helicase. The core archaeal GINS complex contains two subunits that are poorly conserved homologues of the eukaryotic GINS subunits, in complex with a protein containing a domain homologous to the DNA-binding domain of bacterial RecJ. Interaction studies show that archaeal GINS interacts directly with the heterodimeric core primase. Our data suggest that GINS is important in coordinating the architecture of the replication fork and provide a mechanism to couple progression of the MCM helicase on the leading strand with priming events on the lagging strand.

Control of DNA replication: regulation and activation of eukaryotic replicative helicase, MCM.

DNA replication is a key event of cell proliferation and the final target of signal transduction induced by growth factor stimulation. It is also strictly regulated during the ongoing cell cycle so that it occurs only once during S phase and that all the genetic materials are faithfully duplicated. DNA replication may be arrested or temporally inhibited due to a varieties of internal and external causes. Cells have developed intricate mechanisms to cope with the arrested replication forks to minimize the adversary effect on the stable maintenance of genetic materials. Helicases play a central role in DNA replication. In eukaryotes, MCM (minichromosome maintenance) protein complex plays essential roles as a replicative helicase. MCM4-6-7 complex possesses intrinsic DNA helicase activity which translocates on single-stranded DNA form 3' to 5'. Mammalian MCM4-6-7 helicase and ATPase activities are specifically stimulated by the presence of thymine-rich single-stranded DNA sequences onto which it is loaded. The activation appears to depend on the thymine content of this single-strand, and sequences derived from human replication origins can serve as potent activators of the MCM helicase. MCM is a prime target of Cdc7 kinase, known to be essential for activation of replication origins. We will discuss how the MCM may be activated at the replication origins by template DNA, phosphorylation, and interaction with other replicative proteins, and will present a model of how activation of MCM helicase by specific sequences may contribute to selection of replication initiation sites in higher eukaryotes.

Modulation of DNA synthesis in Saccharomyces cerevisiae nuclear extract by DNA polymerases and the origin recognition complex.

We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol alpha, the catalytic subunit of pol delta, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of polepsilon. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, alpha and delta, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.

Recruitment of the ArgR/Mcm1p repressor is stimulated by the activator Gcn4p: a self-checking activation mechanism.

Transcription of the arginine biosynthetic gene ARG1 is repressed by the ArgR/Mcm1p complex in arginine-replete cells and activated by Gcn4p, a transcription factor induced by starvation for any amino acid. We show that all four subunits of the arginine repressor are recruited to ARG1 by Gcn4p in cells replete with arginine but starved for isoleucine/valine. None of these proteins is recruited to the Gcn4p target genes ARG4 and SNZ1, which are not regulated by ArgR/Mcm1p. Mcm1p and Arg80p were found in a soluble complex lacking Arg81p and Arg82p, and both Mcm1p and Arg80p were efficiently recruited to ARG1 in wild-type cells in the presence or absence of exogenous arginine, and also in arg81Delta cells. By contrast, the recruitment of Arg81p and Arg82p was stimulated by exogenous arginine. These findings suggest that Gcn4p constitutively recruits an Mcm1p/Arg80p heterodimer and that efficient assembly of a functional repressor also containing Arg81p and Arg82p occurs only in arginine excess. By recruiting an arginine-regulated repressor, Gcn4p can precisely modulate its activation function at ARG1 according to the availability of arginine.

Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development.

Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes. Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases. Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication. In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis. Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase). Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis. Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals. Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells. Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development.

Conserved homeodomain proteins interact with MADS box protein Mcm1 to restrict ECB-dependent transcription to the M/G1 phase of the cell cycle.

Two homeodomain proteins, Yox1 and Yhp1, act as repressors at early cell cycle boxes (ECBs) to restrict their activity to the M/G1 phase of the cell cycle in budding yeast. These proteins bind to Mcm1 and to a typical homeodomain binding site. The expression of Yox1 is periodic and directly correlated with its binding to, and repression of, ECB activity. The absence of Yox1 and Yhp1 or the constitutive expression of Yox1 leads to the loss of cell-cycle regulation of ECB activity. Therefore, the cell-cycle-regulated expression of these repressors defines the interval of ECB-dependent transcription. Twenty-eight genes, including MCM2-7, CDC6, SWI4, CLN3, and a number of genes required during late M phase have been identified that are coordinately regulated by this pathway.

The role of nucleosome positioning in repression by the yeast alpha 2/Mcm1p repressor.

In Saccharomyces cerevisiae, alpha 2/Mcm1p represses a-cell specific genes. Previous studies indicated that repression by alpha 2/Mcm1p is associated with organization of positioned nucleosomes in promoters of a-cell specific genes such as STE6 and BAR1. Here, we examined the role of nucleosome positioning in repression of STE6-lacZ by alpha 2/Mcm1p. We showed that insertion of an A34 sequence, which is shown to disrupt an array of positioned nucleosomes, affects STE6-lacZ expression. Furthermore, artificial recruitment of TBP is facilitated by the insertion of A34 to derepress STE6-lacZ expression. These findings support the model that nucleosome positioning plays a regulatory role in repression by alpha 2/Mcm1p.

A potential role for mini-chromosome maintenance (MCM) proteins in initiation at the dihydrofolate reductase replication origin.

Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-beta, ori-beta', and ori-gamma) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells.