GENOMICS 54, 424–436 (1998) ARTICLE NO. GE985587 cDNA Cloning and Gene Mapping of Human Homologs for Schizosaccharomyces pombe rad17, rad1, and hus1 and Cloning of Homologs from Mouse, Caenorhabditis elegans, and Drosophila melanogaster Frank B. Dean,*,1 Lubing Lian,† and Mike O’Donnell*,‡ *The Rockefeller University and ‡The Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10021; and †Myriad Genetics, Inc., 390 Wakara Way, Salt Lake City, Utah 84108 Received July 13, 1998; accepted September 17, 1998 INTRODUCTION Mutations in DNA repair/cell cycle checkpoint genes can lead to the development of cancer. The cloning of The progression of a eukaryotic cell through the human homologs of yeast DNA repair/cell cycle check- stages of the cell cycle can be arrested if the events of point genes should yield candidates for human tumor the previous stage of the cell cycle, such as DNA rep- suppressor genes as well as identifying potential tar- lication, have not been completed or if the DNA has gets for cancer therapy. The Schizosaccharomyces sustained some type of damage. The controls on cell pombe genes rad17, rad1, and hus1 have been identi- cycle progression are termed checkpoints (Hartwell fied as playing roles in DNA repair and cell cycle and Weinert, 1989), and they are able to detect checkpoint control pathways. We have cloned the whether the processes of the individual stages of the cDNA for the human homolog of S. pombe rad17, cell cycle have been completed and whether the DNA is RAD17, which localizes to chromosomal location 5q13 intact or in need of repair. Cells that are mutated in by fluorescence in situ hybridization and radiation one of the cell cycle checkpoint genes, however, are able hybrid mapping; the cDNA for the human homolog of to proceed from one stage of the cell cycle to the next S. pombe rad1, RAD1, which maps to 5p14–p13.2; and even if the cellular processes of that stage are incom- the cDNA for the human homolog of S. pombe hus1, plete or in the presence of DNA damage. The G2 phase HUS1, which maps to 7p13–p12. The human gene loci of the cell cycle lies between S phase, in which DNA have previously been identified as regions containing replication takes place, and M phase, when mitosis tumor suppressor genes. In addition, we report the occurs. Thus the G2 checkpoint is critical for ensuring cloning of the cDNAs for genes related to S. pombe that mitosis does not occur until the necessary steps of rad17, rad9, rad1, and hus1 from mouse, Caenorhab- DNA replication, DNA repair, and chromosome dupli- ditis elegans, and Drosophila melanogaster. These in- cation are complete. clude Rad17 and Rad9 from D. melanogaster, hpr-17 Many checkpoint-deficient mutants have been iden- and hpr-1 from C. elegans, and RAD1 and HUS1 from tified in the budding yeast Saccharomyces cerevisiae mouse. The identification of homologs of the S. pombe and in the fission yeast Schizosaccharomyces pombe. rad checkpoint genes from mammals, arthropods, and Genes that link mitosis to the completion of DNA rep- nematodes indicates that this cell cycle checkpoint lication have been isolated (Enoch and Nurse, 1990; pathway is conserved throughout eukaryotes. © 1998 Enoch et al., 1992; McFarlane et al., 1997). In addition, Academic Press many genes that function in DNA repair have been identified as G2 checkpoint control genes (Nasim and Smith, 1975; Al-Khodairy and Carr, 1992; Al-Khodairy Sequence data from this article have been deposited with the et al., 1994), including S. cerevisiae RAD9 (Weinert and EMBL/GenBank Data Libraries under Accession Nos. AF076838, Hartwell, 1990), S. cerevisiae MEC3 (Weinert et al., AF076839, AF076840, AF076841, AF076842, AF076843, AF076844, 1994), S. pombe rad1 (Rowley et al., 1992), S. pombe AF076845, AF076846, AF090170, G41776, G41777, and G41778. rad3 (Jimenez et al., 1992; Bentley et al., 1996), S. 1 To whom correspondence should be addressed at The Rockefeller University, 1230 York Avenue, Box 228, New York, NY 10021. Tele- pombe rad9 (Murray et al., 1991), S. pombe rad17 phone: (212) 327-7255. Fax: (212) 327-7253. E-mail: deanfb@mod. (Griffiths et al., 1995), S. pombe hus1 (Kostrub et al., rockefeller.edu. 1997), and the fungus Ustilago maydis REC1 (Onel et 424 0888-7543/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. HUMAN RAD CHECKPOINT HOMOLOGS 425 al., 1996). The S. pombe rhp9 gene is a homolog of S. pombe rad3 (Savitsky et al., 1995; reviewed by Enoch cerevisiae RAD9 (Willson et al., 1997). Recent studies and Norbury, 1995; Lehmann and Carr, 1995; Jackson, have begun to indicate what the in vivo role of the S. 1996). These proteins have protein kinase activity and cerevisiae checkpoint genes may be (Lydall and Wein- are involved in generating a signal to halt progression ert, 1995). The Rad24, Rad17, and Mec3 proteins ap- through the cell cycle in response to DNA damage. pear to activate an exonuclease activity in vivo while Identification of the human homologs of the yeast G2 the Rad9 protein appears to modulate exonuclease ac- cell cycle checkpoint genes should yield attractive can- tivity. A number of reviews summarize this work (Shel- didates for novel human tumor suppressor genes. The drick and Carr, 1993; Lydall and Weinert, 1996; Stew- human genes are likely to play a cell cycle regulatory art and Enoch, 1996; Carr, 1997). role in human cells, and previously identified human The gene for S. pombe rad17 has been described cell cycle checkpoint genes have been identified as tu- (Griffiths, 1995), as has the gene for its homolog in S. mor suppressors. A human homolog of S. pombe rad9 cerevisiae, RAD24 (Lydall and Weinert, 1997). Cloning was described recently (Lieberman et al., 1996). of S. pombe rad17 revealed that it has an ATP binding We describe here the cDNA cloning of the human site and has extensive homology over its entire length homologs of the S. pombe rad17, rad1, and hus1 genes. to the DNA polymerase accessory proteins known as We have mapped the chromosomal location of the hu- clamp loaders (Griffiths et al., 1995). Clamp loaders man genes and find that the map positions of RAD17 (for instance human RFC, Escherichia coli g complex) and RAD1 correlate with loci for human tumor sup- couple hydrolysis of ATP to positioning a protein ring pressor genes. In addition, we have cloned cDNAs for around duplex DNA. The protein ring tethers the rep- some of the homologs of these genes from mouse, Dro- licase to DNA for high processivity in chromosomal sophila melanogaster, and Caenorhabditis elegans. Re- replication (Kelman and O’Donnell, 1995). The homol- ports of the cloning of human RAD17, RAD1, and ogy to clamp loading subunits suggests that S. pombe HUS1 appeared while this work was in progress rad17 may carry out a similar clamp loading or unload- (Parker et al., 1998a, b; Kostrub et al., 1998). ing function in the DNA repair pathway. Interestingly, while the S. pombe rad17 gene carries out two roles, DNA repair and cell cycle checkpoint regulation, the MATERIALS AND METHODS two functions are separable. Specific point mutations of Isolation of human RAD17. Two sequences were identified in the rad17 that reduced DNA repair activity but did not EST database for human cDNA clones (Accession No. T10666, clone affect checkpoint control were generated. The cloning hbc863, and Accession No. AA133547, clone 586844). Clone hbc863 of the gene for S. pombe rad1 has been described in a was kindly sent by Graeme Bell, The University of Chicago. Clone pair of reports (Sunnerhagen et al., 1990; Long et al., 586844 (IMAGE, Integrated Molecular Analysis of Genomes and Their Expression) was obtained from Genome Systems, Inc. Rapid 1994). The DNA repair and checkpoint functions of amplification of cDNA ends was carried out using Marathon-Ready rad1 are also separable; mutations that have differen- cDNA (Clontech, Palo Alto, CA) as a template. Primer R1702 tial effects on the two activities have been isolated (59-GCGGGATCCCTATGTCCCATCACTCTCGTAGTCTTC-39) an- (Kanter-Smoler et al., 1995). The cloning of its S. cer- nealed to the 39 end of the open reading frame and primed synthesis evisiae homolog, RAD17, has also been described of the antisense strand. Primer AP1 (Clontech) annealed to the Marathon cDNA adaptor product strand at the 59 end of the cDNA (Siede et al., 1996). Extensive work has been performed and primed synthesis of the sense strand. The PCR amplification on its homolog in U. maydis, REC1 (Holliday et al., was performed for 30 cycles of 94°C for 30 s and 68°C for 7 min in a 1976; Holden et al., 1989; Tsukuda et al., 1989). An reaction volume of 50 ml containing 40 mM Tricine–KOH, pH 9.2 at exonuclease activity is associated with the protein 25°C, 15 mM KOAc, 3.5 mM Mg(OAc)2,75mg/ml bovine serum (Thelen et al., 1994), and, again, the gene plays roles in albumin, 200 mM each dNTP, 10 pmol each primer AP1 and R1702, 0.5 ng Marathon-Ready cDNA template, 1 ml Advantage KlenTaq both DNA repair and cell cycle regulation (Onel et al., Polymerase Mix (Clontech). Amplified DNA products (15 ml) were 1995, 1996). The cloning of S. pombe hus1 was de- analyzed by electrophoresis through a 1.0% agarose gel, and a band scribed recently (Kostrub et al., 1997). Yeast strains of DNA 2.6 kb in size was observed. A second PCR amplification was disrupted in hus1 are viable, but are checkpoint-defec- performed under conditions identical to those of the first reaction, tive. with the exception that the template used was 0.5 ng of PCR product of the first reaction, and 10 pmol of primer AP2 (Clontech) was used The occurrence of mutations in the checkpoint con- instead of primer AP1.