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Human and Mouse Homologs of Schizosaccharomyces Pombe Rad1+ and Saccharomyces Cerevisiae RAD17: Linkage to Checkpoint Control and Mammalian Meiosis

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Human and Mouse Homologs of Schizosaccharomyces Pombe Rad1+ and Saccharomyces Cerevisiae RAD17: Linkage to Checkpoint Control and Mammalian Meiosis Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Human and mouse homologs of Schizosaccharomyces pombe rad1+ and Saccharomyces cerevisiae RAD17: linkage to checkpoint control and mammalian meiosis Raimundo Freire,1, Jose R. Murguı´a,2 Madalina Tarsounas,3 Noel F. Lowndes,2 Peter B. Moens,3 and Stephen P. Jackson1,4 1Wellcome Trust/Cancer Research Campaign Institute of Cancer and Developmental Biology, and Department of Zoology, Cambridge University, Cambridge CB2 1QR; 2Imperial Cancer Research Fund, Clare Hall Laboratories, Herts EN6 3LD, UK; 3Department of Biology, York University, Downsview, Ontario M3J 1P3, Canada Preventing or delaying progress through the cell cycle in response to DNA damage is crucial for eukaryotic cells to allow the damage to be repaired and not incorporated irrevocably into daughter cells. Several genes involved in this process have been discovered in fission and budding yeast. Here, we report the identification of human and mouse homologs of the Schizosaccharomyces pombe DNA damage checkpoint control gene rad1+ and its Saccharomyces cerevisiae homolog RAD17. The human gene HRAD1 is located on chromosome 5p13 and is most homologous to S. pombe rad1+. This gene encodes a 382-amino-acid residue protein that is localized mainly in the nucleus and is expressed at high levels in proliferative tissues. This human gene significantly complements the sensitivity to UV light of a S. pombe strain mutated in rad1+. Moreover, HRAD1 complements the checkpoint control defect of this strain after UV exposure. In addition to functioning in DNA repair checkpoints, S. cerevisiae RAD17 plays a role during meiosis to prevent progress through prophase I when recombination is interrupted. Consistent with a similar role in mammals, Rad1 protein is abundant in testis, and is associated with both synapsed and unsynapsed chromosomes during meiotic prophase I of spermatogenesis, with a staining pattern distinct from that of the recombination proteins Rad51 and Dmc1. Together, these data imply an important role for hRad1 both in the mitotic DNA damage checkpoint and in meiotic checkpoint mechanisms, and suggest that these events are highly conserved from yeast to humans. [Key Words: DNA damage; checkpoint control; synaptonemal complex; meiosis; cell cycle; DNA repair] Received May 19, 1998; revised version accepted June 30, 1998. Repair of damage to the genetic material is crucial for tion takes place (Elledge 1996; Paulovich et al. 1997). living organisms because they are continuously exposed The available evidence indicates that specialized protein to DNA-damaging agents, both physiological and envi- complexes sense the DNA damage and trigger inhibitory ronmental. In higher eukaryotes, for example, deficient signals that impinge on the cell cycle machinery. Many DNA repair can lead to tumorigenesis and has been of the genes for these proteins were identified in fission linked to certain degenerative disease states. Eukaryotic and budding yeast through mutations that cause defec- cells respond to DNA damage by delaying progress tive cell cycle checkpoints and hypersensitivity to DNA through the cell cycle and by repairing different types of damaging agents. Conservation of some of these genes in damage with distinct sets of proteins. This cell cycle distantly divergent yeasts indicates that DNA damage delay is called the DNA damage checkpoint and either checkpoint mechanisms are highly conserved through- causes irreversible withdrawal from the cell cycle (Di out evolution (for review, see Lehmann 1996). Leonardo et al. 1994), or allows the cell time to repair the Three key yeast checkpoint genes are Saccharomyces damaged DNA before it is replicated or mitotic segrega- cerevisiae RAD17 (scRAD17; Lydall and Weinert 1995; Siede et al. 1996) and its Schizosaccharomyces pombe homolog rad1+ (sprad1+; Al-Khodairy and Carr 1992; 4Corresponding author. Rowley et al. 1992; Long et al. 1994), scRAD24 and its E-MAIL [email protected]; FAX (01223) 334089. homolog sprad17+ (Griffiths et al. 1995), and sprad3+ and 2560 GENES & DEVELOPMENT 12:2560–2573 © 1998 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/98 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press HRAD1, a human DNA-damage checkpoint gene its homologs scMEC1 and scTEL1 (Morrow et al. 1995; breaks (Storlazzi et al. 1995; Keeney et al. 1997). The Paulovich and Hartwell 1995; Bentley et al. 1996). In subsequent steps in this process require a group of pro- addition to being involved in the DNA damage check- teins involved in DNA mismatch repair or homologous point, sprad1+ is also involved in another cell cycle recombination (Shinohara and Ogawa 1995; Hassold checkpoint triggered in the presence of S-phase inhibi- 1996; Schwacha and Kleckner 1997). Rad51 and Dmc1, tors (the S–M phase checkpoint; Al-Khodairy and Carr two eukaryotic homologs of the bacterial recombination 1992; Rowley et al. 1992). The products of scRAD17 and factor RecA, are found in distinct foci in synapsed mei- sprad1+ show homology to Ustilago maydis Rec1p, a otic chromosomes during stages of prophase I progres- protein implicated in recombination, mutagenesis, and sion in humans, rodents, and budding yeast, suggesting checkpoint control (Onel et al. 1996). Although the pre- that they play important roles in meiotic recombination cise roles of these three factors in the DNA-damage or its signaling (Bishop 1994; Barlow et al. 1997; Moens checkpoint is unclear, Rec1 has been reported to possess et al. 1997). During meiotic recombination in S. cerevi- 38 → 58 exonuclease activity (Thelen et al. 1994). Re- siae, a checkpoint control results in prophase I arrest if cently, spRad1 was found to interact with another DNA recombination is incomplete (Bishop et al. 1992). Several damage checkpoint protein, spHus1, supporting the hy- mitotic DNA damage checkpoint genes (scRAD24, pothesis that these two proteins form part of a multisub- scRAD17, and scMEC1) are involved in this process (Ly- unit DNA damage checkpoint complex (Kostrub et al. dall et al. 1996), revealing important connections be- 1998). tween the DNA damage and meiotic checkpoint con- Strains deficient in scRAD24/sprad17+ have similar trols. Furthermore, mutations in scMEC1 lead to defec- phenotypes to scRAD17/sprad1+-deficient strains, and tive meiosis (Kato and Ogawa 1994), and mutations in the products of these genes display homology with rep- mei-41, the Drosophila homolog of sprad3+/scMEC1, lication factor C subunits (Griffiths et al. 1995), which cause reduced meiotic recombination frequencies and are involved in recruitment of replication factors onto aberrant recombination nodules—the sites of recombi- primed templates during the initiation of DNA replica- nation events (Hari et al. 1995). Recent results indicate tion. Recently, scMec1p, scTel1p, and spRad3 have re- that ATM and ATR also function in the control of mei- ceived much interest because they belong to a family of otic progression. Thus, ATM-deficient humans have re- large proteins that are homologous to phosphatidylino- duced fertility, ATM-deficient mice are infertile as a re- sitol 3-kinase (Jackson 1995; Hoekstra 1997). This family sult of a failure to carry out meiotic prophase I during includes other proteins involved in DNA repair or check- spermatogenesis (Xu et al. 1996; Barlow et al. 1998), and point controls such as the human DNA-dependent pro- ATM and ATR were repoted to be located in synapsed tein kinase catalytic subunit (DNA-PKcs; Hartley et al. and unsynapsed meiotic chromosomes, respectively 1995) and ATR/FRP1, which is the mammalian homolog (Keegan et al. 1996; Barlow et al. 1998). hChk1, the hu- of spRad3 and scMec1p (Bentley et al. 1996; Cimprich et man homolog of the S. pombe checkpoint protein Chk1, al. 1996). Notably, human ATR complements partially is also located along unsynapsed chromosomes, and a the UV sensitivity of a mec1 mutant S. cerevisiae strain possible interaction with ATR has been suggested (Bentley et al. 1996) and recently was shown to encode a (Flaggs et al. 1997). Taken together, these findings imply component of the mammalian G2–M phase DNA dam- a dual role for certain proteins in DNA damage-induced age checkpoint apparatus (Cliby et al. 1998). Also in this mitotic checkpoints and in meiotic checkpoint control family of proteins is the human homolog of scTel1p, mechanisms. It will, therefore, be of great interest and ATM (Savitsky et al. 1995), which functions in the G1–S, importance to identify and characterize other proteins S–M, and G2–M checkpoints that are triggered after ex- that function in these pathways. In this report, we de- posure to ionizing radiation (Lavin and Shiloh 1997). scribe the isolation of mammalian homologs of sprad1+. Other DNA damage checkpoint components that have We show that the human gene complements substan- been identified in mammalian systems are p53 and some tially both the radiation sensitivity and checkpoint phe- of its downstream effectors, such as p21 and p16 (Levine notypes of a sprad1 mutant strain. Furthermore, we find 1997; Shapiro et al. 1998). In addition, hRad9, the human that expression of mammalian Rad1 is high in prolifer- homolog of the S. pombe checkpoint protein Rad9 has ating tissues and is transcriptionally up-regulated in tes- been identified (Lieberman et al. 1996). hRad9 partially tis. Finally, we report that Rad1 is associated with dis- complements the checkpoint defect of a S. pombe rad9 crete foci on the chromosomes of mouse spermatocytes mutant strain after exposure to ionizing radiation (Li- undergoing meiotic prophase I. eberman et al. 1996), supporting the notion that the fun- damentals of checkpoint control are conserved from yeast to humans. Results Recently, a link has been established between the mi- Isolation and chromosomal location of human totic DNA damage checkpoints and control of progres- and mouse homologs of sprad1+ sion through meiosis. During meiotic prophase I, ho- mologous chromosomes pair, synapse, recombine, then By screening expressed-sequence tag (EST) databases for segregate (for a review, see Roeder 1997).
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