Corrado Santocanale

A Mec1-and Rad53-dependent checkpoint controls late-firing origins of DNA replication

DNA replication in eukaryotic cells initiates from many replication origins which fire throughout the S phase of the cell cycle in a predictable pattern: some origins fire early, others late. Little is known about how the initiation of DNA replication and the elongation of newly synthesized DNA strands are coordinated during S phase. Here we show that, in budding yeast, hydroxyurea, which blocks the progression of replication forks from early-firing origins, also inhibits the firing of late origins. These late origins are maintained in the initiation-competent prereplicative state for extended periods. The block to late origin firing is an active process and is defective in yeast with mutations in the rad53 and mec1 checkpoint genes, indicating that regulation of late origin firing may also be an important component of the ‘intra-S-phase’ checkpoint and may aid cell survival under adverse conditions.

ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae.

We have developed a genomic footprinting protocol which allows us to examine protein‐DNA interactions at single copy chromosomal origins of DNA replication in the budding yeast Saccharomyces cerevisiae. We show that active replication origins oscillate between two chromatin states during the cell cycle: an origin recognition complex (ORC)‐dependent post‐replicative state and a Cdc6p‐dependent pre‐replicative state. Furthermore, we show that both post‐ and pre‐replicative complexes can form efficiently on closely apposed replicators. Surprisingly, ARS301 which is active as an origin on plasmids but not in its normal chromosomal location, forms ORC‐ and Cdc6p‐dependent complexes in both its active and inactive contexts. Thus, although ORC and Cdc6p are essential for initiation, their binding is not sufficient to dictate origin use.

Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.

Cdc7 inhibition reveals a p53-dependent replication checkpoint that is defective in cancer cells

Cdc7 is an evolutionarily conserved kinase that regulates S phase by promoting replication origin activation. Down-regulation of Cdc7 by small interfering RNA in a variety of tumor cell lines causes an abortive S phase, leading to cell death by either p53-independent apoptosis or aberrant mitosis. Unlike replication fork blockade, Cdc7-depleted tumor cells do not elicit a robust checkpoint response; thus, inhibitory signals preventing additional cell cycle progression are not generated. In normal fibroblasts, however, a p53-dependent pathway actively prevents progression through a lethal S phase in the absence of sufficient Cdc7 kinase. We show that in this experimental system, p53 is required for the lasting maintenance of this checkpoint and for cell viability. With this work we reveal and begin to characterize a novel mechanism that regulates DNA synthesis in human cells, and we suggest that inhibition of Cdc7 kinase represents a promising approach for the development of a new generation of anticancer agents.

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity.

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.

Dbf4p, an Essential S Phase-Promoting Factor, Is Targeted for Degradation by the Anaphase-Promoting Complex

ABSTRACT The Dbf4p/Cdc7p protein kinase is essential for the activation of replication origins during S phase. The catalytic subunit, Cdc7p, is present at constant levels throughout the cell cycle. In contrast, we show here that the levels of the regulatory subunit, Dbf4p, oscillate during the cell cycle. Dbf4p is absent from cells during G1and accumulates during the S and G2 phases. Dbf4p is rapidly degraded at the time of chromosome segregation and remains highly unstable during pre-Start G1 phase. The rapid degradation of Dbf4p during G1 requires a functional anaphase-promoting complex (APC). Mutation of a sequence in the N terminus of Dbf4p which resembles the cyclin destruction box eliminates this APC-dependent degradation of Dbf4p. We suggest that the coupling of Dbf4p degradation to chromosome separation may play a redundant role in ensuring that prereplicative complexes, which assemble after chromosome segregation, do not immediately refire.

EVIDENCE FOR A CDC6P-INDEPENDENT MITOTIC RESETTING EVENT INVOLVING DNA POLYMERASE ALPHA

C.Desdouets and C.Santocanale contributed equally to this work

Cdc7 Kinase Inhibitors : Pyrrolopyridinones as Potential Antitumor Agents. 1. Synthesis and Structure-Activity Relationships

Cdc7 kinase is an essential protein that promotes DNA replication in eukaryotic organisms. Genetic evidence indicates that Cdc7 inhibition can cause selective tumor-cell death in a p53-independent manner, supporting the rationale for developing Cdc7 small-molecule inhibitors for the treatment of cancers. In this paper, the synthesis and structure-activity relationships of 2-heteroaryl-pyrrolopyridinones, the first potent Cdc7 kinase inhibitors, are described. Starting from 2-pyridin-4-yl-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one, progress toward a simple scaffold, tailored for Cdc7 inhibition, is reported.

Drf1, a novel regulatory subunit for human Cdc7 kinase

Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs. Cdc7, an evolutionarily conserved serine–threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins. Binding of the cell cycle‐regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity. This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells. Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4. Drf1 is a nuclear cell cycle‐regulated protein, it binds to Cdc7 and activates the kinase. Therefore, human Cdc7, like cyclin‐dependent kinases, can be activated by alternative regulatory subunits. Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome.

Cdc7 Is an Active Kinase in Human Cancer Cells Undergoing Replication Stress

Cdc7 kinase promotes and regulates DNA replication in eukaryotic organisms. Multiple mechanisms modulating kinase activity in response to DNA replication stress have been reported, supporting the opposing notions that Cdc7 either plays an active role under these conditions or, conversely, is a final target inactivated by a checkpoint response. We have developed new immnunological reagents to study the properties of human Cdc7 kinase in cells challenged with the ribonucleotide reductase inhibitor hydroxyurea or with the DNA topoisomerase II inhibitor etoposide. We show that Cdc7·Dbf4 and Cdc7·Drf1 complexes are stable and active in multiple cell lines upon drug treatment, with Cdc7·Dbf4 accumulating on chromatin-enriched fractions. Cdc7 depletion by small interfering RNA in hydroxyurea and etoposide impairs hyper-phosphorylation of Mcm2 at specific Cdc7-dependent phosphorylation sites and drug-induced hyper-phosphorylation of chromatin-bound Mcm4. Furthermore, sustained inhibition of Cdc7 in the presence of these drugs increases cell death supporting the notion that the Cdc7 kinase plays a role in maintaining cell viability during replication stress.