Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms.

The stable propagation of genetic information requires that the entire genome of an organism be faithfully replicated once and only once each cell cycle. In eukaryotes, this replication is initiated at hundreds to thousands of replication origins distributed over the genome, each of which must be prohibited from re-initiating DNA replication within every cell cycle. How cells prevent re-initiation has been a long-standing question in cell biology. In several eukaryotes, cyclin-dependent kinases (CDKs) have been implicated in promoting the block to re-initiation, but exactly how they perform this function is unclear. Here we show that B-type CDKs in Saccharomyces cerevisiae prevent re-initiation through multiple overlapping mechanisms, including phosphorylation of the origin recognition complex (ORC), downregulation of Cdc6 activity, and nuclear exclusion of the Mcm2-7 complex. Only when all three inhibitory pathways are disrupted do origins re-initiate DNA replication in G2/M cells. These studies show that each of these three independent mechanisms of regulation is functionally important.     

DNA stretching by bacterial initiators promotes replication origin opening

Many replication initiators form higher-order oligomers that process host replication origins to promote replisome formation. In addition to dedicated duplex-DNA-binding domains, cellular initiators possess AAA+ (ATPases associated with various cellular activities) elements that drive functions ranging from protein assembly to origin recognition. In bacteria, the AAA+ domain of the initiator DnaA has been proposed to assist in single-stranded DNA formation during origin melting. Here we show crystallographically and in solution that the ATP-dependent assembly of Aquifex aeolicus DnaA into a spiral oligomer creates a continuous surface that allows successive AAA+ domains to bind and extend single-stranded DNA segments. The mechanism of binding is unexpectedly similar to that of RecA, a homologous recombination factor, but it differs in that DnaA promotes a nucleic acid conformation that prevents pairing of a complementary strand. These findings, combined with strand-displacement assays, indicate that DnaA opens replication origins by a direct ATP-dependent stretching mechanism. Comparative studies reveal notable commonalities between the approach used by DnaA to engage DNA substrates and other, nucleic-acid-dependent, AAA+ systems.     

Replication fork movement sets chromatin loop size and origin choice in mammalian cells

Genome stability requires one, and only one, DNA duplication at each S phase. The mechanisms preventing origin firing on newly replicated DNA are well documented, but much less is known about the mechanisms controlling the spacing of initiation events(2,3), namely the completion of DNA replication. Here we show that origin use in Chinese hamster cells depends on both the movement of the replication forks and the organization of chromatin loops. We found that slowing the replication speed triggers the recruitment of latent origins within minutes, allowing the completion of S phase in a timely fashion. When slowly replicating cells are shifted to conditions of fast fork progression, although the decrease in the overall number of active origins occurs within 2 h, the cells still have to go through a complete cell cycle before the efficiency specific to each origin is restored. We observed a strict correlation between replication speed during a given S phase and the size of chromatin loops in the next G1 phase. Furthermore, we found that origins located at or near sites of anchorage of chromatin loops in G1 are activated preferentially in the following S phase. These data suggest a mechanism of origin programming in which replication speed determines the spacing of anchorage regions of chromatin loops, that, in turn, controls the choice of initiation sites.     

Role for a Drosophila Myb-containing protein complex in site-specific DNA replication

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-beta, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-beta (refs 7-9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.     

Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin

Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented.     

CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing

To maintain genome stability, DNA replication is strictly regulated to occur only once per cell cycle. In eukaryotes, the presence of 'licensing proteins' at replication origins during the G1 cell-cycle phase allows the formation of the pre-replicative complex. The removal of licensing proteins from chromatin during the S phase ensures that origins fire only once per cell cycle. Here we show that the CUL-4 ubiquitin ligase temporally restricts DNA-replication licensing in Caenorhabditis elegans. Inactivation of CUL-4 causes massive DNA re-replication, producing cells with up to 100C DNA content. The C. elegans orthologue of the replication-licensing factor Cdt1 (refs 2, 3) is required for DNA replication. C. elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease during S phase and instead remain constant in the re-replicating cells. Removal of one genomic copy of cdt-1 suppresses the cul-4 re-replication phenotype. We propose that CUL-4 prevents aberrant re-initiation of DNA replication, at least in part, by facilitating the degradation of CDT-1.     

Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control

Cyclin-dependent kinases (CDKs) limit the activation of DNA replication origins to once per cell cycle by preventing the assembly of pre-replicative complexes (pre-RCs) during S, G2 and M phases of the cell cycle in the budding yeast Saccharomyces cerevisiae. CDKs inhibit each pre-RC component (ORC, Cdc6, Cdt1/Mcm2-7) by different mechanisms. We show here that the mitotic CDK, Clb2/Cdc28, binds tightly to an amino-terminal domain (NTD) of Cdc6, and that Cdc6 in this complex is unable to assemble pre-RCs. We present evidence indicating that this Clb2-dependent mechanism contributes to preventing re-replication in vivo. CDK interaction with the NTD of Cdc6 is mediated by the cyclin subunit Clb2, and could be reconstituted with recombinant Clb2 protein and synthetic NTD peptides. Tight Clb2 binding occurred only when the NTD was phosphorylated on CDK consensus sites. Human CDKs containing cyclins A, B and E also bound specifically to phospho-NTD peptides. We propose that direct binding of cyclins to phosphopeptide motifs may be a widespread phenomenon contributing to the targeting of CDKs to substrates.     

Reduction of the minimal sequence for initiation of DNA synthesis by qualitative or quantitative changes of an initiator protein

Initiation of DNA synthesis at an origin of DNA replication involves complex protein-DNA interactions that are still poorly understood. Some of these interactions are highly specific and involve proteins (initiator proteins) thought to be essential for regulation of the initiation process because of their rate-limiting activity. We show here that both qualitative and quantitative changes in one of these proteins have profound effects on protein-DNA interactions at an origin of DNA replication, and are sufficient to reduce to less than one-third the minimal sequence required for initiation. The general implications of these findings are discussed.     

Chromosome length influences replication-induced topological stress

During chromosome duplication the parental DNA molecule becomes overwound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression, this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replication-induced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of Saccharomyces cerevisiae chromosomes. This highlights the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter, chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin-related Smc5/6 complex. The frequency of chromosomal association sites of the Smc5/6 complex increases in response to chromosome lengthening, chromosome circularization, or inactivation of topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings that form behind the replication machinery.     

Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication

Cell-free replication systems for simian virus 40 (SV40) DNA are taken to be a model for the replication of eukaryotic chromosomes, because only one viral protein is required to supplement the replication proteins provided by a human cell extract. To prove that these cellular proteins function in chromosomal DNA replication we have begun to identify homologous proteins in an organism that can be genetically manipulated. Here we report the identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae and show that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication. Yeast RF-A, a multi-subunit phosphoprotein, is similar to the human protein in its chromatographic behaviour, subunit structure and DNA-binding activity. The yeast protein will fully substitute for the human protein in an early stage of the initiation of SV40 DNA replication. Substitution of yRF-A in the complete SV40 replication system, however, results in reduced DNA replication, presumably due to a requirement for species-specific interactions between yeast RF-A and the DNA polymerase complex.     

S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex

The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.     

Structure and mechanism of the chromatin remodelling factor ISW1a

Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolutions of 3.25?? and 3.60??, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a 'protein ruler'.     

Effect of novobiocin on initiation of DNA replication in Bacillus subtilis.

The initiation of DNA replication of small replicons in vitro involves conformational changes in the whole DNA molecule or in the region near to the replication origin. One striking finding has been the role of DNA gyrase (that is, the necessity for supercoiled structure) in the initial stage of ColE1 replication in vitro. However, little is known about the effect of gyrase on the initiation of replication of bacterial chromosomes in vivo. We have constructed a map of cleavage sites of restriction enzymes at the region of the origin of replication of the Bacillus subtilis chromosome (accompanying paper). This has now enabled us to examine the effect of novobiocin, a selective inhibitor of DNA gyrase, on the replication of the specific chromosomal segments near the origin and to seek a possible role for the gyrase in the initiation of chromosomal replication. We have found that only a limited segment of the chromosome at the origin region was replicated in the presence of novobiocin. This effect allowed us to locate the site of the origin of replication to within a DNA fragment of molecular weight 3.4 x 10(6).     

XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.