C. elegans CUL-4 prevents rereplication by promoting the nuclear export of CDC-6 via a CKI-1-dependent pathway

Genome stability requires that genomic DNA is replicated only once per cell cycle. The replication-licensing system ensures that the formation of prereplicative complexes is temporally separated from the initiation of DNA replication [1-4]. The replication-licensing factors Cdc6 and Cdt1 are required for the assembly of prereplicative complexes during G1 phase. During S phase, metazoan Cdt1 is targeted for degradation by the CUL4 ubiquitin ligase [5-8], and vertebrate Cdc6 is translocated from the nucleus to the cytoplasm [9, 10]. However, because residual vertebrate Cdc6 remains in the nucleus throughout S phase [10-13], it has been unclear whether Cdc6 translocation to the cytoplasm prevents rereplication [1, 2, 14]. The inactivation of C. elegans CUL-4 is associated with dramatic levels of DNA rereplication [5]. Here, we show that C. elegans CDC-6 is exported from the nucleus during S phase in response to the phosphorylation of multiple CDK sites. CUL-4 promotes the phosphorylation and subsequent translocation of CDC-6 via negative regulation of the CDK-inhibitor CKI-1. Rereplication can be induced by coexpression of nonexportable CDC-6 with nondegradable CDT-1, indicating that redundant regulation of CDC-6 and CDT-1 prevents rereplication. This demonstrates that CDC-6 translocation is critical for preventing rereplication and that CUL-4 independently controls both replication-licensing factors.     

Cdt1 proteolysis is promoted by dual PIP degrons and is modulated by PCNA ubiquitylation

Cdt1 plays a critical role in DNA replication regulation by controlling licensing. In Metazoa, Cdt1 is regulated by CRL4(Cdt2)-mediated ubiquitylation, which is triggered by DNA binding of proliferating cell nuclear antigen (PCNA). We show here that fission yeast Cdt1 interacts with PCNA in vivo and that DNA loading of PCNA is needed for Cdt1 proteolysis after DNA damage and in S phase. Activation of this pathway by ultraviolet (UV)-induced DNA damage requires upstream involvement of nucleotide excision repair or UVDE repair enzymes. Unexpectedly, two non-canonical PCNA-interacting peptide (PIP) motifs, which both have basic residues downstream, function redundantly in Cdt1 proteolysis. Finally, we show that poly-ubiquitylation of PCNA, which occurs after DNA damage, reduces Cdt1 proteolysis. This provides a mechanism for fine-tuning the activity of the CRL4(Cdt2) pathway towards Cdt1, allowing Cdt1 proteolysis to be more efficient in S phase than after DNA damage.     

Regulation of germline stem cell proliferation downstream of nutrient sensing

In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs) at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC) and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks) and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1) functional and SCF^Skp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2) replication-coupled proteolysis mediated by the Cullin4-DDB1^Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed.     

Replication: DNA building block synthesis on demand

Correct regulation of DNA nucleotide biosynthesis is emerging as a key issue of importance for genome integrity. The fission yeast Spd1 protein can modulate the activity of ribonucleotide reductase (RNR) by at least three different mechanisms. Now a paper reports that Spd1 turnover is linked to ongoing DNA synthesis.     

The Schizosaccharomyces pombe replication inhibitor Spd1 regulates ribonucleotide reductase activity and dNTPs by binding to the large Cdc22 subunit

Ribonucleotide reductase (RNR) is an essential enzyme that provides the cell with a balanced supply of deoxyribonucleoside triphosphates for DNA replication and repair. Mutations that affect the regulation of RNR in yeast and mammalian cells can lead to genetic abnormalities and cell death. We have expressed and purified the components of the RNR system in fission yeast, the large subunit Cdc22p, the small subunit Suc22p, and the replication inhibitor Spd1p. It was proposed (Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M., and Caspari, T. (2003) Genes Dev. 17, 1130-1140) that Spd1 is an RNR inhibitor, acting by anchoring the Suc22p inside the nucleus during G1 phase. Using in vitro assays with highly purified proteins we have demonstrated that Spd1 indeed is a very efficient inhibitor of fission yeast RNR, but acting on Cdc22p. Furthermore, biosensor technique showed that Spd1p binds to the Cdc22p with a KD of 2.4 microM, whereas the affinity to Suc22p is negligible. Therefore, Spd1p inhibits fission yeast RNR activity by interacting with the Cdc22p. Similar to the situation in budding yeast, logarithmically growing fission yeast increases the dNTP pools 2-fold after 3 h of incubation in the UV mimetic 4-nitroquinoline-N-oxide. This increase is smaller than the increase observed in budding yeast but of the same order as the dNTP pool increase when synchronous Schizosaccharomyces pombe cdc10 cells are going from G1 to S-phase.     

A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells

Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.     

Recombinant Cdt1 induces rereplication of G2 nuclei in Xenopus egg extracts

A crucial regulation for maintaining genome integrity in eukaryotes is to limit DNA replication in S phase to only one round. Several models have been proposed; one of which, the licensing model, predicted that formation of the nuclear membrane restricts access to chromatin to a positive replication factor. Cdt1, a factor binding to origins and recruiting the MCM2-7 helicase, has been identified as a component of the licensing system in Xenopus and other eukaryotes. Nevertheless, evidence is missing demonstrating a direct role for unscheduled Cdt1 expression in promoting illegitimate reinitiation of DNA synthesis. We show here that Xenopus Cdt1 is absent in G2 nuclei, suggesting that it might be either degraded or exported. Recombinant Cdt1, added to egg extracts in G2, crosses the nuclear membrane, binds to chromatin, and relicenses the chromosome for new rounds of DNA synthesis in combination with chromatin bound Cdc6. The mechanism involves rebinding of MCM3 to chromatin. Reinitiation is blocked by geminin only in G2 and is not stimulated by Cdc6, demonstrating that Cdt1, but not Cdc6, is limiting for reinitiation in egg extracts. These results suggest that removal of Cdt1 from chromatin and its nuclear exclusion in G2 is critical in regulating licensing and that override of this control is sufficient to promote illegitimate firing of origins.     

Expression of Cdc18/Cdc6 and Cdt1 during G2 phase induces initiation of DNA replication

Cdc18/Cdc6 and Cdt1 are essential initiation factors for DNA replication. In this paper we show that expression of Cdc18 in fission yeast G2 cells is sufficient to override the controls that ensure one S phase per cell cycle. Cdc18 expression in G2 induces DNA synthesis by re-firing replication origins and recruiting the MCM Cdc21 to chromatin in the presence of low levels of Cdt1. However, when Cdt1 is expressed together with Cdc18 in G2, cells undergo very rapid, uncontrolled DNA synthesis, accumulating DNA contents of 64C or more. Our data suggest that Cdt1 may potentiate re-replication by inducing origins to fire more persistently, possibly by stabilizing Cdc18 on chromatin. In addition, low level expression of a mutant form of Cdc18 that cannot be phosphorylated by cyclin-dependent kinases is not sufficient to induce replication in G2, but does so only when co-expressed with Cdt1. Thus, regulation of both Cdc18 and Cdt1 in G2 plays a crucial role in preventing the re-initiation of DNA synthesis until the next cell cycle.     

Chromatin remodeler sucrose nonfermenting 2 homolog (SNF2H) is recruited onto DNA replication origins through interaction with Cdc10 protein-dependent transcript 1 (Cdt1) and promotes pre-replication complex formation

From late mitosis to the G(1) phase of the cell cycle, ORC, CDC6, and Cdt1 form the machinery necessary to load MCM2-7 complexes onto DNA. Here, we show that SNF2H, a member of the ATP-dependent chromatin-remodeling complex, is recruited onto DNA replication origins in human cells in a Cdt1-dependent manner and positively regulates MCM loading. SNF2H physically interacted with Cdt1. ChIP assays indicated that SNF2H associates with replication origins specifically during the G(1) phase. Binding of SNF2H at origins was decreased by Cdt1 silencing and, conversely, enhanced by Cdt1 overexpression. Furthermore, SNF2H silencing prevented MCM loading at origins and moderately inhibited S phase progression. Although neither SNF2H overexpression nor SNF2H silencing appeared to impact rereplication induced by Cdt1 overexpression, Cdt1-induced checkpoint activation was inhibited by SNF2H silencing. Collectively, these data suggest that SNF2H may promote MCM loading at DNA replication origins via interaction with Cdt1 in human cells. Because efficient loading of excess MCM complexes is thought to be required for cells to tolerate replication stress, Cdt1- and SNF2H-mediated promotion of MCM loading may be biologically relevant for the regulation of DNA replication.     

Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry.     

Novel mutator mutants of E. coli nrdAB ribonucleotide reductase: insight into allosteric regulation and control of mutation rates

Ribonucleotide reductase (RNR) is the enzyme critically responsible for the production of the 5'-deoxynucleoside-triphosphates (dNTPs), the direct precursors for DNA synthesis. The dNTP levels are tightly controlled to permit high efficiency and fidelity of DNA synthesis. Much of this control occurs at the level of the RNR by feedback processes, but a detailed understanding of these mechanisms is still lacking. Using a genetic approach in the bacterium Escherichia coli, a paradigm for the class Ia RNRs, we isolated 23 novel RNR mutants displaying elevated mutation rates along with altered dNTP levels. The responsible amino-acid substitutions in RNR reside in three different regions: (i) the (d)ATP-binding activity domain, (ii) a novel region in the small subunit adjacent to the activity domain, and (iii) the dNTP-binding specificity site, several of which are associated with different dNTP pool alterations and different mutational outcomes. These mutants provide new insight into the precise mechanisms by which RNR is regulated and how dNTP pool disturbances resulting from defects in RNR can lead to increased mutation.