Scientific Report 2005

Molecular Biology

Cell-Cycle Checkpoints, DNA Repair, and Oxidative Stress Response

P. Russell, C. Chahwan, S. Coulon, L.-L. Du, P.-H. Gaillard, V. Martin, T. Nakamura, C. Noguchi, E. Noguchi, M. Rodriguez, P. Shanahan, K. Tanaka, H. Zhao

The cellular responses to DNA damage and cytotoxic stress are highly conserved through evolution. A fortunate consequence of this conservation is that “simple” eukaryotes such as the fission yeast Schizosaccharomyces pombe can be used as model systems for more complex multicellular organisms. We use S pombe to study cell-cycle checkpoints, DNA repair, and stress response mechanisms. Defects in these mechanisms underlie a number of human diseases, including cancer.

DNA Replication Checkpoint

The challenging task of replicating a eukaryotic genome is often made more difficult by conditions that interfere with progression of the replisome, the complex formed by the close association of the key proteins used during DNA replication. Protein complexes bound to DNA, chemical adducts in DNA, and deoxyribonucleotide starvation are among the situations that can impede replisomes. The DNA replication checkpoint senses stalled replication forks and directs cellular responses that help preserve the integrity of the genome. One of these responses is the S-M checkpoint. This checkpoint delays the onset of mitosis (M phase) while DNA synthesis (S phase) is under way, thereby providing time to recover from stalled forks. The same checkpoint also controls how damaged DNA is replicated.

DNA-dependent protein kinases, such as ATM and ATR in humans and Rad3 in fission yeast, are central components of the replication checkpoint. Acting in conjunction with regulatory subunits (e.g., Rad26 in fission yeast) and other protein complexes, these kinases activate checkpoint effector kinases. The effector of the replication checkpoint in fission yeast is Cds1 (Chk2). A few years ago, we discovered mediator of replication checkpoint-1 (Mrc1), an adaptor or mediator protein that directs the replication checkpoint signal from Rad3 to Cds1. We recently discovered that the forkhead-associated domain of Cds1 mediates the binding of Cds1 to Mrc1. This interaction allows Rad3 to activate Cds1.

Cds1 controls repair systems that are required to tolerate stalled replication forks. We hope to better understand these systems by identifying proteins that associate with the forkhead-associated domain of Cds1. Mus81, a novel protein related to the XPF nucleotide excision repair protein, was identified in a screen for such proteins. We found that Mus81 associates with another protein, Eme1, to form a structure-specific endonuclease that resolves X-shaped Holliday junctions. In recent studies with T. Wang, Stanford University, Stanford, California, and M.N. Boddy, Department of Molecular Biology, we discovered that phosphorylation of Mus81 by Cds1 helps preserve genome integrity when replication forks arrest. We hypothesize that the phosphorylation prevents the Mus81-Eme1 complex from cleaving stalled replication forks.

Stalled forks are potentially unstable structures prone to rearrangement and collapse. We previously reported that the protein Swi1 helps preserve stalled forks and is necessary for strong activation of Cds1. Recent studies with J.R. Yates, Department of Cell Biology, indicated that Swi1 associates with Swi3 to form a fork-protection complex. We found that the complex travels with the replisome during DNA replication. It is therefore ideally placed to detect, stabilize, and signal stalled replication forks (Fig. 1). We speculate that Swi1 and Swi3 homologs in humans have equivalent functions.

Fig. 1. Stabilization of stalled replication forks. The fork-protection complex (FPC), which consists of Swi1 and Swi3, travels with the replisome. Mrc1 also appears to travel with the fork. When the replisome stalls at obstructions in the fork or for other reasons, the fork-protection complex and Mrc1 are required for activation of Cds1 by Rad3-Rad26 kinase. The Rad9-Rad1-Hus1 (9-1-1) complex is also required for Cds1 activation.

DNA Damage Checkpoint

The DNA damage checkpoint prevents the onset of mitosis when DNA is damaged (Fig. 2). This checkpoint is enforced by the protein kinase Chk1, which is activated by Rad3. Activation of Chk1 requires the adaptor protein Crb2. Crb2 is rapidly recruited to double-stranded breaks in DNA. We recently found that Rad3 and Tel1 (the ATM homolog in fission yeast) stimulate Crb2 recruitment by phosphorylating histone H2A at the DNA break site. We also found that the tandem C-terminal BRCT domains in Crb2 are essential for Crb2 homo-oligomerization.

Fig. 2. The unicellular yeast S pombe divides by medial fission (top left panel). It has 3 chromosomes and approximately 4000 genes. The DNA damage checkpoint arrests division in cells exposed to ionizing radiation (+IR) (top right panel). Pulse-field gel electrophoresis shows that the chromosomes are fragmented by 120 Gy of ionizing radiation (bottom panel). About 3 hours are required to repair the DNA, necessitating a checkpoint that prevents mitosis while DNA repair is under way.

Recently, we investigated how Tel1/ATM is recruited for sites of DNA damage and how it is activated. These studies, done in collaboration with T. Hunter, the Salk Institute, La Jolla, California, revealed that Tel1/ATM interacts with the extreme C terminus of Nbs1. Nbs1 is a subunit of the Mre11-Rad50-Nbs1 complex that associates with and processes double-stranded breaks. We found that the interaction with Nbs1 is essential for ATM activation.

Oxidative Stress Response

Oxidative stress caused by reactive oxygen species can be highly toxic, causing damage to proteins, lipids, and nucleic acids. Oxidative stress elicits a complex gene expression response that is orchestrated in large part by MAP kinase cascades. The fission yeast Spc1 MAP kinase pathway is homologous to the p38 pathway in humans. We recently discovered Csx1, a protein that collaborates with Spc1 to control gene expression in response to oxidative stress. Csx1 is an RNA-binding protein that mediates global control of gene expression in response to oxidative stress by binding and stabilizing mRNA that encodes Atf1, a transcription factor that is also regulated by Spc1. Most recently, we focused on a newly discovered family of proteins that interact with Csx1.

Publications

Du, L.L., Moser, B.A., Russell, P. Homo-oligomerization is the essential function of the tandem BRCT domains in the checkpoint protein Crb2. J. Biol. Chem. 279:38409, 2004.

Kai, M., Boddy, M.N., Russell, P., Wang, T.S.F. Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress. Genes Dev. 19:919, 2005.

McGowan, C.H., Russell, P. The DNA damage response: sensing and signaling. Curr. Opin. Cell Biol. 16:629, 2004.

Nakamura, T.M., Moser, B.A., Du, L.L., Russell, P. Cooperative control of Crb2 by ATM-family and Cdc2 kinases is essential for the DNA damage checkpoint in fission yeast. Mol. Cell. Biol., in press.

Noguchi, E., Noguchi, C., McDonald, W.H., Yates, J.R. III, Russell, P. Swi1 and Swi3 are components of a replication fork protection complex in fission yeast. Mol. Cell. Biol. 24:8342, 2004.

Tanaka, K., Russell, P. Cds1 phosphorylation by Rad3-Rad26 kinase is mediated by forkhead-associated domain interaction with Mrc1. J. Biol. Chem. 279:32079, 2004.

You, Z., Chahwan, C., Bailis, J., Hunter, T. Russell, P. ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1. Mol. Cell. Biol. 25:5363, 2005.

Zhao, H., Russell, P. DNA binding domain in the replication checkpoint protein Mrc1 of Schizosaccharomyces pombe. J. Biol. Chem. 279:53023, 2004.