Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51⁺ from Schizosaccharomyces pombe

Abstract

The Schizosaccharomyces pombe DNA repair gene rhp51؉ encodes a RecA-like protein with the DNAdependent ATPase activity required for homologous recombination. The level of the rhp51 ؉ transcript is increased by a variety of DNA-damaging agents. Its promoter has two cis-acting DNA damage-responsive elements (DREs) responsible for DNA damage inducibility. Here we report identification of Rdp1, which regulates rhp51؉ expression through the DRE of rhp51 ؉ . The protein contains a zinc finger and a polyalanine tract similar to ones previously implicated in DNA binding and transactivation or repression, respectively. In vitro footprinting and competitive binding assays indicate that the core consensus sequences (NGG/TTG/A) of DRE are crucial for the binding of Rdp1. Mutations of both DRE1 and DRE2 affected the damage-induced expression of rhp51 ؉ , indicating that both DREs are required for transcriptional activation. In addition, mutations in the DREs significantly reduced survival rates after exposure to DNA-damaging agents, demonstrating that the damage response of rhp51 ؉ enhances the cellular repair capacity. Surprisingly, haploid cells containing a complete rdp1 deletion could not be recovered, indicating that rdp1؉ is essential for cell viability and implying the existence of other target genes. Furthermore, the DNA damage-dependent expression of rhp51 ؉ was significantly reduced in checkpoint mutants, raising the possibility that Rdp1 may mediate damage checkpoint-dependent transcription of rhp51All organisms have developed defense mechanisms to respond to genotoxic materials causing genetic injury. One response to DNA damage or DNA synthesis inhibition is to delay the cell cycle by blocking DNA replication and/or mitotic division. Another is the transcriptional induction of several genes whose products may contribute to DNA repair capacity (22).During past decades, such damage-inducible genes have been identified and partially characterized for bacteria, yeasts, and higher eukaryotes (22). In particular, a large number of genes are induced in response to DNA damage and/or inhibition of DNA replication in Saccharomyces cerevisiae (22,49). These include RNR2 (which encodes a small subunit of ribonucleotide reductase [19,28] [12]), which are involved in DNA repair. However, the biological significance of the transcriptional induction of these genes has been uncovered only recently. A study has revealed that Dun1p serine/threonine protein kinase is involved in the transcriptional activation of RNR2 and has delineated a pathway by which the damage signal is transduced to a checkpoint and transcription response apparatus (65). The repressor protein Crt1p was found to bind to X boxes on the RNR2 and RNR3 promoters and to mediate repression of the genes by cooperating with the Tup1p-Ssn6p corepressor. DNA damage-induced hyperphosphorylation of Crt1p enables the protein to dissociate from X boxes, which leads to derepression of RNR2 transcription. This dissociation is also dependent on the MEC1-RAD53-DUN1 damage-signal...

Global Gene Expression Responses of Fission Yeast to Ionizing Radiation

“…Microarray analysis has recently identified a Mec1-dependent gene expression signature in response to DNAdamaging agents that includes a large set of genes responsive to diverse environmental stresses (Gasch et al, 2001). A role for the DNA damage checkpoint pathway in regulating transcriptional responses to DNA-damaging agents has also been identified in fission yeast (Harris et al, 1996;Shim et al, 2000), although it is not known to what extent the checkpoint pathway coordinates damage-induced transcription in this microorganism.…”

“…In lower eukaryotes, studies with the budding yeast Saccharomyces cerevisiae have identified a large number of damageinduced genes, including many involved in DNA metabolism and DNA repair (Aboussekhra et al, 1996;Gasch et al, 2001). In the distantly related fission yeast Schizosaccharomyces pombe, rhp51 ϩ , uvi15 ϩ , uvi31 ϩ , UVDE ϩ , and rhp16 ϩ have been shown to be DNA damage inducible (Bang et al, 1996;Davey et al, 1997;Shim et al, 2000).…”

“…Microarray analysis has recently identified a Mec1-dependent gene expression signature in response to DNAdamaging agents that includes a large set of genes responsive to diverse environmental stresses (Gasch et al, 2001). A role for the DNA damage checkpoint pathway in regulating transcriptional responses to DNA-damaging agents has also been identified in fission yeast (Harris et al, 1996;Shim et al, 2000), although it is not known to what extent the checkpoint pathway coordinates damage-induced transcription in this microorganism.In fission yeast, the Sty1/Spc1 stress-kinase is closely related to the mammalian and Drosophila JNK and p38 stresskinase pathways, and to the Hog1 pathway in S. cerevisiae . In contrast to the Hog1 pathway in budding yeast, Sty1/Spc1 is activated in response to a wide variety of environmental stresses, including UV, methylmethane sulfonate (MMS), oxidative stress, osmotic stress, and heatshock (Millar et al, 1995;Shiozaki and Russell, 1995;Degols et al, 1996;Degols and Russell, 1997;Shieh et al, 1997).…”

“…In lower eukaryotes, studies with the budding yeast Saccharomyces cerevisiae have identified a large number of damageinduced genes, including many involved in DNA metabolism and DNA repair (Aboussekhra et al, 1996;Gasch et al, 2001). In the distantly related fission yeast Schizosaccharomyces pombe, rhp51 ϩ , uvi15 ϩ , uvi31 ϩ , UVDE ϩ , and rhp16 ϩ have been shown to be DNA damage inducible (Bang et al, 1996;Davey et al, 1997;Shim et al, 2000).DNA damage checkpoint pathways function to delay the eukaryotic cell cycle in response to DNA damage, thus providing an opportunity for DNA repair. Central to the DNA damage checkpoint pathway are two highly conserved phosphoinositol-related kinases, Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) and their yeast homologs SpTel1 and SpRad3 in S. pombe, and ScTel1 and ScMec1 in S. cerevisiae.…”

Global gene expression of fission yeast in response to cisplatin

“…In the wt strain we found up-regulation of the recombinational repair gene rhp51 (homolog to RAD51 of Saccharomyces cerevisiae) encoding a RecA-like protein whose DNA-dependent ATPase activity is required for homologous recombination. Thus, in S. pombe, recombinational repair is triggered by DDP as shown for a variety of DNA-damaging agents that increase the levels of the rhp51 transcript [34]. A putative damage-inducible deoxycytidyl transferase (SPBC1347.01c) possibly involved in mutagenic translesion DNA synthesis, was also induced by DDP.…”