Scientific Report 2007

Molecular and Experimental Medicine

Division of Molecular Oncology

Checking on DNA During Replication

X. Wu, A.Y.-L. Lee, C. Nievera, E. Olson, T. Chiba

Genome instability is a hallmark of the malignant phenotype and a driving force for tumorigenesis. S phase is genetically the most vulnerable period of the cell cycle. In this phase, DNA must be replicated faithfully in a timely fashion, and the entire genome must be duplicated exactly once per cell cycle. Cell-cycle checkpoints have evolved to monitor the integrity of the eukaryotic genome and ensure the completion of DNA replication and the repair of DNA damage before progression of the cycle.

In one area of our research, we focus on a disease-linked complex termed Mre11/Rad50/Nbs1 (MRN), which participates in multiple pathways to maintain genome stability. In humans, hypomorphic mutations in the genes NBS1 and MRE11 lead to Nijmegen breakage syndrome and ataxia-telangiectasia–like disorder, respectively. Cells from patients with Nijmegen breakage syndrome or ataxia-telangiectasia–like disorder have a defect in the intra-S-phase checkpoint, but the molecular mechanisms are unclear.

Recently, we found that MRN directly interacts with replication protein A in unperturbed cells and that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with replication protein A leads to pronounced radioresistant DNA synthesis, without affecting phosphorylation of Nbs1 or structural maintenance of chromosomes protein 1 after ionizing radiation. Moreover, MRN is recruited to sites at or adjacent to replication origins by replication protein A and acts there to inhibit new origin firing upon ionizing radiation. These findings suggest that MRN at sites proximal to the origin of chromosomal replication has a direct role in controlling the initiation of DNA replication in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.

The second focus of our research is understanding how DNA replication is controlled so that DNA is replicated once and only once per cell cycle. Rereplication of the genome, or even a segment of it, could lead to genome instability. We found that the S-phase checkpoint mediated by the ataxia telangiectasia–mutated and Rad3-related (ATR) pathway acts as a surveillance mechanism to prevent rereplication, so that disruption of licensing control by the overexpression of the licensing factor Cdt1 does not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. Single-stranded DNA accumulated by uncontrolled DNA unwinding mediated by mini-chromosome maintenance due to Cdt1 overexpression is the initial signal to activate the checkpoint. Our studies reveal the molecular mechanisms by which the ATR-mediated S-phase checkpoint pathway prevents DNA rereplication and thus increases our understanding of how rereplication is prevented in mammalian cells.

Publications

Olson, E., Nievera, C.J., Klimovich, V., Fanning, E., Wu, X. RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint. J. Biol. Chem. 281:39517, 2006.

Olson, E., Nievera, C.J., Liu, E., Lee, A.Y., Chen, L., Wu, X. The Mre11 complex mediates the S-phase checkpoint through an interaction with RPA. Mol. Cell. Biol., in press.

Olson, E., Nievera, C.J., Liu, E., Lee, A.Y., Chen, L., Wu, X. The Mre11/Rad50/Nbs1 complex acts both upstream and downstream of ATR to regulate the S-phase checkpoint following UV treatment. J. Biol. Chem., in press.