Chromosomal DNA Replication and Cancer Research

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In human cells, genomic DNA, which carries genetic information, has to be replicated faithfully, completely, and only once during a single cell cycle to maintain integrity. If some errors occur during copying DNA (DNA replication), then it would sometimes lead to bad consequences. "Cancer" is one and serious example resulting from such replication errors (mutations).

Molecular mechanisms for cell cycle regulation of DNA replication initiation
Cdt1, a central factor for the cell cycle regulation of replication initiation: Elucidating the strict regulations by three ubiquitin ligases
Cdt1 deregulation induces chromosomal instability, a mechanism leading to cancer
Cdt1-geminin system could be a novel molecular target for anti-cancer chemotherapeutic agents

Research Projects

Molecular mechanisms for cell cycle regulation of DNA replication initiation
Several molecular mechanisms contribute to the maintenance of genomic integrity. Replicative DNA polymerases synthesize new daughter strand using complementary parental strand. However, DNA polymerases very rarely incorporate incorrect nucleotides. The mismatch repair pathway that removes inappropriate nucleotides is a fail-safe mechanism for such errors and the disturbance is well known to cause genomic instability and eventual cancer. Chromosomal DNAs are often damaged, for example by ultraviolet, which should also be repaired. Disruption of such repair mechanism also leads to cancer.

We have been interested in elucidating molecular mechanisms for cell cycle regulation of DNA replication initiation, another crucial aspect of replication controls. In human cells, genomic DNA is fragmented into multiple chromosomes, which may allow genome size to expand during the evolution. As a result, DNA replication initiates from multiple replication origins. However, effective operation of the "multiple replication origin" system gives rise to an important problem: i.e. multiple replication origins should each be activated precisely only once during each S phase.

Recent research progress by us and other groups has uncovered the mechanisms (for review, see Cell Div, 1:22, 2006). It is now clear that the "once and only once replication per single cell cycle" is achieved by the periodic assembly and disassembly of pre-replication complexes (pre-RCs) at replication origins (Fig.1). The pre-RC assembly reaction, known as "licensing", involves the loading of a presumptive replicative helicase, the MCM2-7 complex, onto chromatin by the origin recognition complex (ORC), CDC6 and Cdt1. Two critical inhibitory factors for the pre-RC assembly are cyclin/Cdks (Cdk1 and Cdk2) and geminin. During late mitosis through the G1 phase, a cell cycle regulatory E3 ubiquitin ligase APC/C restrains cyclins and geminin by targeting them for proteolysis through polyubiquitination. Thus, pre-RC assembly only occurs during this period (Fig. 1A). Following APC/C inactivation at the onset of S phase, Cdks are activated, stimulating DNA unwinding by MCM. Then, DNA polymerases synthesize new DNA (Fig.1B).


Fig. 1. A model for cell cycle regulation of pre-RC assembly

To prevent re-replication, the re-establishment of pre-RC, in other words re-binding of MCM, needs to be suppressed during the S, G2 and M phases of the cell cycle (Fig. 1B). Cdks play a central role also in this context by preventing re-establishment of pre-RC through multiple mechanisms. One is by phosphorylation of CDC6, leading to CDC6 nuclear export (Fig. 2). ORC1 is degraded after S phase, presumably depending on phosphorylation by cyclin A/Cdks and binding to SCFSkp2 ubiquitin ligase.


Fig. 2. Nuclear export of CDC6 during the S phase

Cdt1, a central factor for the cell cycle regulation of replication initiation: Elucidating the strict regulations by three ubiquitin ligases
It was originally suggested that inhibition of Cdt1 function after S phase is due to geminin binding. However, we have recently demonstrated that three ubiquitin ligases strictly control Cdt1 proteolysis, showing that Cdt1 is a central player in the cell cycle regulation of replication initiation (Fig. 1B and 3). During S and G2 phases, Cdt1 is brought to proteolysis by Cdk phosphorylation-dependent SCFSkp2-mediated ubiquitination (J. Biol. Chem., 279: 19691-7, 2004). Interestingly, Cdt1 is also regulated by replication-coupled, Cullin4-DDB1Cdt2 ubiquitin ligase-mediated ubiquitination, which is dependent on Cdt1 binding to PCNA, an eukaryotic sliding clamp stimulating DNA polymerases (EMBO J., 25: 1126-36, 2006). In addition, when cells enter quiescence, Cdt1 is rapidly cleared by APC/CCdh1-mediated proteolysis (Mol. Biol. Cell, doi: 10.1091/mbc.E07-09-0859, 2007).


Fig. 3. Molecular mechanisms for Cdt1 proteolytic regulation by three ubiquitin ligases

Cdt1 deregulation induces chromosomal instability, a mechanism leading to cancer
As expected from the strict regulation, deregulation of Cdt1 is a deleterious insult, leading to re-replication and/or chromosomal damage (Fig. 4; J. Cell Sci., 119: 3128-40, 2006; Mol. Biol. Cell, doi: 10.1091/mbc.E07-09-0859, 2007). The induced chromosomal instability may eventually lead to carcinogenesis and Cdt1 overexpression is in fact often observed in human cancers (J. Cell Sci., 119: 3128-40, 2006). By other groups, it has been suggested that Cdt1 overexpression could endow cells with the transforming ability.


Fig.4. Large nuclei resulting from massive re-replication induced by a degradation-resistant mutant Cdt1

Cdt1-geminin system could be a novel molecular target for anti-cancer chemotherapeutic agents
We also think that tumor cells could be selectively eliminated by modulating the Cdt1-geminin interactions and have been seeking small molecule compounds that affect the interactions (for details, see Biochim. Biophys. Acta, doi: 10.1016/j.bbagen.2007.09.005, 2007).