Fission Yeastrad12⁺Regulates Cell Cycle Checkpoint Control and Is Homologous to the Bloom’s Syndrome Disease Gene

Abstract

The human BLM gene is a member of the Escherichia coli recQ helicase family, which includes the Saccharomyces cerevisiae SGS1 and human WRN genes. Defects in BLM are responsible for the human disease Bloom's syndrome, which is characterized in part by genomic instability and a high incidence of cancer. Here we describe the cloning of rad12 ؉ , which is the fission yeast homolog of BLM and is identical to the recently reported rhq1؉ gene. We showed that rad12 null cells are sensitive to DNA damage induced by UV light and ␥ radiation, as well as to the DNA synthesis inhibitor hydroxyurea. Overexpression of the wild-type rad12 ؉ gene also leads to sensitivity to these agents and to defects associated with the loss of the S-phase and G 2 -phase checkpoint control. We showed genetically and biochemically that rad12 ؉ acts upstream from rad9 ؉ , one of the fission yeast G 2 checkpoint control genes, in regulating exit from the S-phase checkpoint. The physical chromosome segregation defects seen in rad12 null cells combined with the checkpoint regulation defect seen in the rad12 ؉ overproducer implicate rad12 ؉ as a key coupler of chromosomal integrity with cell cycle progression.The fission yeast Schizosaccharomyces pombe undergoes a dose-dependent G 2 delay in response to DNA damage caused by radiation (1, 27). Cells remain arrested at this G 2 checkpoint while DNA damage is repaired, then enter mitosis and resume progression through the cell cycle. Six checkpoint rad genes have been identified in S. pombe: rad1 ϩ , rad3ϩ , rad26 ϩ , and hus1 ϩ (1, 2, 9, 27). Mutations in any of these genes result in almost identical phenotypes. The mutant strains are all sensitive to radiation and to agents which transiently inhibit DNA replication, and all lack the G 2 checkpoint, preventing mitotic entry in the presence of such damage (1, 2, 9, 27). While little is known about G 2 checkpoint genes in humans, homologs of S. pombe rad3 ϩ (4, 5) and rad9 ϩ (19) have recently been isolated. Recent studies have implicated cell cycle gene mutations in causing cancer. It is likely that mutations in genes regulating the G 1 and G 2 checkpoints also have the potential to increase cancer susceptibility.The phenomenon of genomic instability has been firmly established as a crucial step in the genesis of cancer (14,31,33). While genomic instability is seen as a step in the progression of cancer cells, there are also several genetic diseases that lead to genomic instability and, ultimately, to cancer. Among these diseases is the rare autosomal recessive disorder Bloom's syndrome (12). The defective gene in Bloom's syndrome, BLM, encodes a putative DNA helicase that is homologous to the Escherichia coli RecQ and Saccharomyces cerevisiae Sgs1 helicases (11,34). While the BLM and SGS1 proteins contain centrally located helicase domains that are homologous to RecQ, they are much larger than RecQ and show homology, both 5Ј and 3Ј, to the helicase core (26, 35, 37).We describe here the cloning of the fission yeast rad12 ϩ gene, which encodes a helic...

Werner's Syndrome Protein Is Required for Correct Recovery after Replication Arrest and DNA Damage Induced in S-Phase of Cell Cycle

“…The observed sensitivity of Rqh1 and Sgs1 mutants to arrest of replication fork progression proved that repression of illegitimate recombination is essential for fruitful recovery from S-phase arrest (Stewart et al, 1997;Davey et al, 1998;Frei and Gasser, 2000). An anti-hyperrecombinational role of WRN has been proposed in several models (Chakraverty and Hickson, 1999;Shen and Loeb, 2000) and it is consistent with the partial suppression of the sgs1 mutant phenotype by transfection with WRN (Yamagata et al, 1998).…”

“…Such a phenotype, resulting from unequal distribution of the genetic material between the two daughter cells, is also associated with mutations in the Rad genes, which are responsible of the checkpoint response (Enoch and Nurse, 1990). However, yeast defective in the RecQ helicases Rqh1 and Sgs1 do not show an altered checkpoint response after replication arrest (Stewart et al, 1997;Davey et al, 1998;Frei and Gasser, 2000). In fact, it has been demonstrated that the reversible S-phase arrest (i.e., arrest followed by retain of cell viability) also requires protective functions that are distinct from cell cycle checkpoint controls (Stewart et al, 1997).…”

“…Functional interaction between proteins involved in DNA replication, such as replication protein A (RPA) and DNA polymerase ␦, and WRN also have been reported (Shen et al, 1998;Brosh et al, 1999;Kamath-Loeb et al, 2000). Furthermore, in yeast, the RecQlike proteins seem involved in suppression of hyperrecombination, S-phase checkpoint control, and correct DNA segregation (Gangloff et al, 1994, Watt et al, 1995Stewart et al, 1997;Davey et al, 1998;Yamagata et al, 1998;Frei and Gasser, 2000). Noteworthy, yeast mutants in such RecQ-like helicases are extremely sensitive to agents that cause replication arrest, such as hydroxyurea because of uncontrolled illegitimate recombinational events (Stewart et al, 1997;Yamagata et al, 1998).…”

Bloom helicase is involved in DNA surveillance in early S phase in vertebrate cells

“…In S. pombe, Rqh1 mutants show arrested DNA replication and normal cell division in response to hydroxyurea (HU) as well as signi®cant defects in chromosome segregation during subsequent mitosis (Stewart et al, 1997). In S. cerevisiae, Sgs1 mutants are hypersensitive to HU (Yamagata et al, 1998), and they exhibit mitotic hyper-recombination, resulting in increased frequencies of ectopic, interchromosomal homologous and intrachromosomal excision as well as poor sporulation (Davey et al, 1998;Watt et al, 1996). Thus, during interrupted S phase, these yeast RecQ homologues are required to prevent inappropriate recombination, thus regulating genetic exchange and maintaining genomic stability (Davey et al, 1998;Stewart et al, 1997;Watt et al, 1996).…”

“…In S. cerevisiae, Sgs1 mutants are hypersensitive to HU (Yamagata et al, 1998), and they exhibit mitotic hyper-recombination, resulting in increased frequencies of ectopic, interchromosomal homologous and intrachromosomal excision as well as poor sporulation (Davey et al, 1998;Watt et al, 1996). Thus, during interrupted S phase, these yeast RecQ homologues are required to prevent inappropriate recombination, thus regulating genetic exchange and maintaining genomic stability (Davey et al, 1998;Stewart et al, 1997;Watt et al, 1996). In the S. cerevisiae Sgs1 mutant, BLM can suppress increased homologous and illegitimate recombination, restore the increased sensitivity to HU (Yamagata et al, 1998) and prevent premature aging and increased homologous recombination (HR) and the rDNA loci (Heo et al, 1999).…”