Scientific Report 2006

Molecular Biology

Cell-Cycle Checkpoints, DNA Damage, and Oxidative Stress Responses

P. Russell, C. Chahwan, C. Dovey, L.-L. Du, P.-H. Gaillard, V. Martin, B.A. Moser, T.M. Nakamura, M.A. Rodrìguez-Gabriel, J. Williams, Y. Yamada

DNA damage and oxidative stress elicit cellular responses that are highly conserved throughout eukaryotic evolution. Consequently, studies of genetically tractable microorganisms such as the fission yeast Schizosaccharomyces pombe can provide a useful framework for the design and interpretation of experiments with 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.

Checkpoints

The DNA replication and damage checkpoints prevent the onset of mitosis when DNA replication is interrupted or when DNA is damaged. A single double-strand break is sufficient to arrest the cell cycle. One aim of our studies is to understand how cells detect DNA damage and transmit a checkpoint signal that arrests the cell cycle.

Chk1 is the effector kinase of the DNA damage checkpoint. It regulates the activities of Cdc25 and Mik1/Wee1 proteins, which modulate the inhibitory phosphorylation of the cyclin-dependent kinase Cdc2. Chk1 activation by Rad3 requires the adaptor protein Crb2. Crb2 is rapidly recruited to double-strand breaks in DNA. Rad3 and Tel1 (the ATM homolog in fission yeast) stimulate Crb2 recruitment by phosphorylating a serine residue near the C terminus of histone H2A in the vicinity of double-strand breaks.

Our data indicate that tandem C-terminal BRCT domains in Crb2 associate directly with phosphorylated histone H2A. Crb2 recruitment to double-strand breaks also requires the constitutive methylation of lysine at position 20 in histone H4. This step most likely involves a direct interaction with a Tudor motif in Crb2 that is located to the N-terminal side of the BRCT motifs. We recently found that these 2 histone modifications cooperate in a nonredundant mechanism to promote recruitment of Crb2 to double-strand breaks (Fig. 1). Remarkably, neither histone modification is required for recruitment of Crb2 to sustained double-strand breaks that cannot be repaired by homologous recombination. We recently discovered that the histone modification–independent recruitment of Crb2 to double-strand breaks involves association between phosphorylated threonine-215 in Crb2 and another checkpoint protein known as Cut5. In future studies, we will determine whether the mechanisms that regulate Crb2 in fission yeast are conserved for the analogous proteins in human cells.

DNA Repair

Bloom, Warner, and Rothmund-Thomson syndromes in humans are typified by predisposition to cancer or premature aging. These syndromes, which all result from defects in DNA helicases, are characterized by genomic instability arising from inappropriate homologous recombination. To better understand this process, we used a 2-hybrid screen to identify novel proteins that associate with Srs2 DNA helicase in fission yeast.

We discovered a previously uncharacterized protein that promotes the formation of toxic recombination structures in yeast mutants that lack one or more DNA helicases. This protein, which we christened Sws1 because it has a SWIM-type zinc finger, is conserved from yeast to humans. In collaborative mass spectrometry and proteomics studies with J.R. Yates, Department of Cell Biology, we found that Sws1 forms a complex with 2 other proteins known as Rlp1 and Rdl1. Bioinformatic analysis revealed that these 2 proteins are Rad51 paralogs (i.e., homologous sequences derived from gene duplication) that promote the formation of the Rad51 nucleoprotein filament during homologous recombination. Rlp1 and Rdl1 are equivalent to human XRCC2 and RAD51D, proteins implicated in other human diseases characterized by genomic instability. In collaboration with C.H. McGowan, Department of Molecular Biology, we found that using small interfering RNA to silence the gene for human SWS1 reduced the occurrence of homologous recombination structures. Our current studies are aimed at providing a deeper biochemical and structural understanding of the SWS1-XRCC2-RAD51D complex.

Fig. 1. Parallel mechanisms of recruiting the DNA damage checkpoint protein Crb2 to sites of DNA damage. Top, A proposed mechanism of how Crb2 associates with double-strand breaks. One mode of association involves interactions with modified histones. The tandem C-terminal BRCT domains associate with the phosphorylated C-terminal region of histone H2A. The Tudor domain interacts with the constitutive methylation of histone H4 on lysine at position 20. These modes of interaction are not redundant. A third mode of interaction involves the phosphorylated threonine at position 215 (T215) region of Crb2 and the Cut5. Cut5 is proposed to specifically bind to the single-stranded DNA region near the end of the double-strand break. This binding might involve the association of Cut5 with other proteins that bind to single-stranded DNA. Bottom, Fission yeast cells that express Crb2 were tagged with yellow fluorescent protein (YFP) and Cut5 tagged with cyan fluorescent protein (CFP). The cells were engineered to express the HO endonuclease and to contain a single HO cleavage site. The YFP-Crb2 and Cut5-CFP foci indicate large-scale accumulation of these proteins at the site of the double-strand break created by HO endonuclease.

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 overall 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 have focused on a newly discovered family of proteins that interact with Csx1. We have named these proteins Cip1 and Cip2 (for Csx1-interacting proteins 1 and 2). Remarkably, elimination of Cip1 or Cip2 results in substantial recovery of the sensitivity of Csx1 mutant cells to oxidative stress, suggesting that Cip1 and Cip2 are part of a mechanism that degrades Atf1 mRNA.

Publications

Cavero, S., Chahwan, C., Russell, P. Xlf1 is required for DNA repair by nonhomologous end-joining in Schizosaccharomyces pombe. Genetics, in press.

Coulon, S., Noguchi, E., Noguchi, C., Du, L.-L., Nakamura, T.M., Russell, P. Rad22^Rad52-dependent repair of ribosomal DNA repeats cleaved by Slx1-Slx4 endonuclease. Mol. Biol. Cell 17:2081, 2006.

de Bruin, R.A.M., Kalashnikova, T.I., Chahwan, C., McDonald, W.H., Wohlschlegel, J., Yates III, J.R., Russell, P., Wittenberg, C. Constraining G1-specific transcription to late G1-phase: The MBF-associated corepressor Nrm1 acts via negative feedback. Mol. Cell. 23:483, 2006.

Du, L.-L., Nakamura, T.M., Russell, P. Histone modification-dependent and -independent pathways for recruitment of checkpoint protein Crb2 to double-strand breaks. Genes Dev. 20:1583, 2006.

Martin, V., Chahwan, C., Gao, H., Blais, V., Wohlschlegel, J., Yates, J.R. III, McGowan, C.H., Russell, P. Sws1 is a conserved regulator of homologous recombination in eukaryotic cells. EMBO J. 25:2564, 2006.

Martìn, V., Rodrìguez-Gabriel, M.A., McDonald, W.H., Watt, S., Yates, J.R. III, Bähler, J., Russell, P. Cip1 and Cip2 are novel RNA-recognition-motif proteins that counteract Csx1 function during oxidative stress. Mol. Biol. Cell 17:1176, 2006.

Matsumoto, S., Ogino, K., Noguchi, E., Russell, P., Masai, H. Hsk1-Dfp1/Him1, the Cdc7-Dbf4 kinase in Schizosaccharomyces pombe, associates with Swi1, a component of the replication fork protection complex. J. Biol. Chem. 280:42536, 2005.

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. 25:10721, 2005.

Rodrìguez-Gabriel, M.A., Russell, P. Distinct signaling pathways respond to arsenite and reactive oxygen species in Schizosaccharomyces pombe. Eukaryot. Cell 4:1396, 2005.

Rodriguez-Gabriel, M.A., Watt, S., Bähler, J., Russell, P. Upf1, an RNA helicase required for nonsense-mediated mRNA decay, modulates the transcriptional response to oxidative stress in fission yeast. Mol. Cell. Biol. 26:6347, 2006.