MIPALRELGS ATPEEYNTW QKPRQILCQF IDRILTDVNV VAVELVKKTD SQPTSVMLLD FIQHIMKSSP LMFVNVSGSH EAKGSCIEFS NWIITRLLRI AATPSCHLLH KKICEVICSL LFLFKSKSPA IFGVLTKELL QLFEDLVYLH RRNVMGHAVE WPWMSRFLS QLDEHMGYLQ SAPLQLMSMQ NLEFIEVTLL MVLTRIIAIV FFRRQELLLW QIGCVLLEYG SPKIKSLAIS FLTELFQLGG LPAQPASTFF SSFLELLKHL VEMDT

A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Meclp (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function-Mei-41, Meclp, and Rad3-and as such may be the functional human counterpart of these proteins. A rigid ordering of events in the mitotic cell cycle aids in accurate replication and segregation of the chromosomes, two processes necessary but not sufficient for maintenance of genomic integrity (1). Cells are frequently subjected to occurrences that interfere with DNA replication or result in DNA damage and that would be deleterious if not repaired. To ensure the order and completion of events in the cell cycle, a number of regulatory checkpoints exist including ones involved in S phase and in the transition from GI to S and G2 to M. The hydroxyurea-sensitive checkpoint (S/M) prevents cells from exiting S phase until DNA replication is complete, while the DNA damage-regulated checkpoints are capable of slowing the progression of S phase and preventing entry into S phase (Gi/S) or progression through mitosis (G2/M) in the presence of DNA damage, allowing time for its repair (2-4). Working in concert, the DNA repair and checkpoint machinery allow the cell to respond to DNA damage or delays in cell cycle progression, ensuring the fidelity of the genome. As might be anticipated, mutations in the genes involved in the checkpoint machinery can lead to a range of chromosomal aberrations or cancer (5, 6), and by studying these mutant phenotypes in several species, a range of genes has been identified that play a role in checkpoint functions. In Saccharomyces cerevisiae, the essential gene MEC1/ESRI (mitosis entry checkpoint) is required for regulation of the S/M and G2/M checkpoints (7), the rate of ongoing S phase in response to damage (3), and meiotic recombination (8). Cells with mutations in MEC1 (mecl-1) proceed directly to mitosis when The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. DNA replication is inhibited with hydroxyurea and are unable to delay the onset of mitosis (G2/M) on induction of DNA damage. Mei-41 appears to be a structural and functional homolog of MEC1 in Drosophila melanogaster required for monitoring the integrity of the genome at the S/M and G2/M checkpoints (9). Mei-41 mutant cells display high levels of mitotic chromosomal instability and like mecl-1 mutants are deficient in meiotic recombination. The Schizosaccharomyces pombe rad3+ gene is also related to MEC1 and mei-41 in sequence and function (10, 11). t RecentlyATM (ataxia telangiectasia, mutated), a gene encoding a human homolog of Mei-41, Rad3 and Meclp, was identified as the defective gene in individuals with ataxia telangiectasia (AT) (13). AT cells are defective in many of the DNA damage-induced checkpoint functions but appear to be proficient in the replication checkpoint functions (reviewed in ref. 12). The proteins encoded by these genes are members of an emerging family of high molecular weight kinases known as the PIK-related kinases (PIK, phosphatidylinositol kinases) (1416). Other members of this family include the FKBP12rapamycin-associated protein (FRAP) (17, 18), TOR1/DRR1 and TOR2/DRR2 (19-21), the catalytic subunit of DNAdependent protein kinase (DNA-PKcs) (22), and TEL1 (23, 24). While the C-terminal regions of the PIK-related kinases possess a high degree of sequence similarity to the catalytic domain of the previously characterized phosphatidylinositol (PI) 3/4-kinases, they appear to be distinct in both overall sequence and function. We describe here the cloning of a new human member of the PIK-related kinase family that we have named FRP1 (FRAPrelated protein). Three overlapping clones have been obtained from a Jurkat T-cell cDNA library. The 2644-amino acid protein encoded by this open reading frame is highly homologous to the Drosophila Mei-41, S. cerevisiae Meclp, and S. pombe Rad3 proteins. Since FRP1 may by analogy have a cell cycle checkpoint function in mammalian cells, we have mapped the FRP1 gene to its chromosomal locus.t MATERIALS AND METHODS Molecular Cloning. Expressed sequence tags (ESTs) were searched in the data base of ESTs using the NCBI BLAST e-mail Server and the TBLASTN program. M13 single-stranded DNA was isolated from a human Jurkat T-cell AZAP II cDNA Abbreviations: FRAP, FKBP12-rapamycin associated protein; FRP1, FRAP-related protein; MEC1, mitosis entry checkpoint; PIK, phosphatidylinositol kinase; ATM, ataxia telangiectasia, mutated; DNAPKcs, DNA-dependent protein kinase, catalytic subunit; FISH, fluorescence in situ hybridization; EST, expressed sequence tag. *To whom reprint requests should be addressed. tThe published sequence of rad3+ encodes a protein of 1070 amino acids that has been noted to be incomplete at both the N and C termini. The sequence ofrad3+ has been reported to encode a protein of 2386 amino acids with similarity to several members of the PIK-related kinase family (9, 12). Our analysis was performed with the available partial sequence data. 4The sequence reported in this paper has been deposited in the GenBank data base (accession no. U49844).