Identification of a Human Homologue of theSchizosaccharomyces pombe rad17 + Checkpoint Gene*

In the fission yeast Schizosaccharomyces pombe the rad17 + gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human cDNA homologue of the S. pombe rad17 + checkpoint gene, designatedHrad17. Hrad17 has 49% identity to theS. pombe rad17 + sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and in cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates that Hrad17 is located on chromosome 4q13.3–21.2. This region is subject to loss of heterozygosity in several human cancers. To begin to understand the protein-protein interactions of the human checkpoint machinery, we have used the yeast two-hybrid system to examine potential interactions between Hrad1, Hrad9, and Hrad17. We demonstrate a physical interaction between Hrad17 and Hrad1 but no interaction with Hrad9.

In the fission yeast Schizosaccharomyces pombe the rad17 ؉ gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human cDNA homologue of the S. pombe rad17 ؉ checkpoint gene, designated Hrad17. Hrad17 has 49% identity to the S. pombe rad17 ؉ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and in cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates that Hrad17 is located on chromosome 4q13.3-21.2. This region is subject to loss of heterozygosity in several human cancers. To begin to understand the protein-protein interactions of the human checkpoint machinery, we have used the yeast two-hybrid system to examine potential interactions between Hrad1, Hrad9, and Hrad17. We demonstrate a physical interaction between Hrad17 and Hrad1 but no interaction with Hrad9.
Cell cycle checkpoints are regulatory pathways ensuring that the events of the cell cycle are completed with high fidelity and in an orderly fashion (reviewed in Refs. [1][2][3]. When cells are subjected to conditions that interfere with DNA replication or cause damage to DNA, a signal is sent to halt cell cycle progression, thus permitting cell cycle phase completion or DNA repair (1)(2)(3). The loss of checkpoint control in mammalian cells results in genomic instability, leading to the amplification, rearrangement, or loss of chromosomes, events associated with tumor progression (2,4). In Schizosaccharomyces pombe the products of six genes: rad1 ϩ , rad3 ϩ , rad9 ϩ , rad17 ϩ , rad26 ϩ , and hus1 ϩ have been identified as essential components of the checkpoint pathways (5). Several of the S. pombe checkpoint genes have structural homologues in the budding yeast, and further conservation across eukaryotes has recently been demonstrated with the cloning of two human homologues of S. pombe rad3 ϩ , ATM (ataxia telangiectasia mutated) (6) and ATR (ataxia telangiectasia and rad3 ϩ related) (7,8); a human homologue of S. pombe rad9 ϩ , Hrad9 (9); and a human homologue of S. pombe rad1 ϩ , Hrad1 (49).
Currently, little is known about the biochemistry of checkpoint control; however, the identification and characterization of human homologues of yeast checkpoint genes provides clear evidence that checkpoint pathways are conserved between mammals and yeast. The genetic data in yeast suggest that a complex of proteins mediates the monitoring of replicationspecific structures and damaged DNA (10), and recent biochemical studies in yeast and humans suggest that the cell cycle arrest in response to DNA damage is brought about by the activation of a signal transduction pathway involving the protein kinases ATM/ATR and Hchk1 resulting in inhibitory phosphorylation of Cdc25 and subsequent stabilization of the inhibitory Tyr 15 phosphorylation of Cdc2 (7,(11)(12)(13).
The S. pombe rad17 mutant is defective in both the DNA damage-dependent and the DNA replication-dependent checkpoints (14). The rad17 ϩ gene has been cloned (15) and shows significant sequence similarity with Saccharomyces cerevisiae RAD24 and components of mammalian replication factor C, which are required to load the replicative DNA polymerases ␦ and ⑀ onto primed DNA templates (16 -19). Rad17 is not a functional homologue of a replication factor C subunit, although the sequence similarity may reflect some shared biological activity such as association with elements of the replication machinery or binding of specific DNA structures (15).
In this report we describe the cloning and characterization of a novel human cDNA, designated Hrad17, which is highly similar to the S. pombe rad17 ϩ checkpoint gene. We also demonstrate that Hrad17 interacts with the recently identified Hrad1 but not Hrad9.

EXPERIMENTAL PROCEDURES
Cloning and Sequencing of Human rad17-A search for sequences similar to S. pombe rad17 ϩ was carried out using the TBLASTN program (20) against the proprietary LifeSeq® data base (Incyte Pharmaceuticals Inc., Palo Alto, CA) as well as public domain data bases such as GenBank TM . Deduced amino acid sequences were aligned using the CLUSTALW program, and similarity was determined with a blosum62 amino acid substitution matrix. Clones with significant sequence similarity were ordered from Incyte or the Merck/Washington University EST 1 collection, and the complete sequence of their inserts was determined. DNA sequencing was carried out on double-stranded plasmid DNA with dye-terminator chemistry as prescribed by the manufacturer (Perkin-Elmer/Applied Biosystems), and the products were resolved on an ABI Prism TM 377 Automated Sequencer. The complete DNA sequences of relevant inserts were aligned as described. The 5Ј end of the longest cDNA was extended by 5Ј RACE-PCR according to the manufacturer's instructions (CLONTECH). Two gene-specific primers were designed to the 5Ј end of the putative Hrad17 ORF: GSP1(5Ј-TTCTAT-GACTTGAGTTATTCACACC-3Ј) and GSP2 (5Ј-GGCAGTAATAGTA-GAGACGCCAC-3Ј). These were used in a nested PCR reaction with Marathon-Ready human placental cDNA (CLONTECH) as the template. The first PCR reaction made use of the GSP1 PCR primer for the 3Ј end, combined with the AP1 primer from CLONTECH that is com-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address: Cardiovascular Metabolism and Musculoskeletal Research Dept., Zeneca Pharmaceuticals, Alderley Edge, Cheshire, UK.
plementary to an adaptor ligated to the 5Ј end of all Marathon-Ready cDNAs. The second PCR reaction started from 1-5 l of the first and used the GSP2 in combination with a nested AP2 primer (CLONTECH). The reaction conditions for the first RACE-PCR were 30 cycles of 30 s at 95°C, 30 s at 65°C, and 1 min at 72°C. The subsequent nested PCR consisted of two-step cycles, each containing a 30-s denaturation step at 95°C and a combined annealing and elongation step for 2 min, which was carried out at 72°C for the first five cycles, at 70°C for the five subsequent cycles, and at 68°C for the remaining 25 cycles. The reaction products were resolved by agarose gel electrophoresis, and specific reaction products were excised from the gel, purified using the QIA-Quick gel extraction kit (Qiagen) and ligated into the pCR2.1-TOPO vector (Invitrogen). The insert sequence of eight independent clones was determined and compared with the putative Hrad17 partial cDNA sequence. The 5Ј RACE clones extended the partial Hrad17 sequence to a putative initiation codon as determined by alignment with the S. pombe rad17 sequence.
PCR primers were designed to amplify the complete Hrad17 ORF (OAP094, 5Ј-GCCGAATTCATGAATCAGGTAACAGACTGGGTT-3Ј, and OP076, 5Ј-GGCGGATCCTCGAGTCTGTCCCATCACTCTCGTA-GT-3Ј) to enable subcloning into various expression vectors. The complete ORF was PCR-amplified from cDNA prepared from human SK-N-MC neuroblastoma cells using the following PCR reaction conditions: 30 cycles of 30 s at 95°C, 30 s at 64°C, and 2 min at 75°C. The amplification products were directly cloned into the pCR2.1-TOPO vector (Invitrogen), and the DNA sequence from three independent clones of Hrad17 was determined.
Northern Analysis-Two multiple tissue Northern blots (CLON-TECH) and a cancer cell line Northern blot (CLONTECH) were hybridized with a DNA probe corresponding to the complete Hrad17 ORF labeled with [␣-32 P]dCTP by random hexamer priming using the Primea-Gene Kit (Promega). The blots were washed at high stringency (0.1 ϫ SSC, 0.1% SDS, 50°C, 2 ϫ 20 min) and exposed to Kodak X-Omat autoradiography film with intensifying screens at Ϫ70°C. The blots were then rehybridized with a human ␤-actin probe (CLONTECH) labeled with [␣-32 P]dCTP as described above. The films were scanned, and quantification of the bands was performed with the program Image Master 1D (Amersham Pharmacia Biotech).

Identification of a Human Homologue of S. pombe rad17 ϩ -
A human expressed sequence tag cDNA clone (number 515944) was identified in the proprietary LifeSeq® data base (Incyte Pharmaceuticals Inc., Palo Alto, CA) using the TBLASTN homology searching program (20) with the S. pombe rad17 ϩ amino acid sequence as the query. This clone was purchased from Incyte Pharmaceuticals and DNA sequence analysis of the 1.9-kb insert revealed an incomplete ORF that was highly similar to the S. pombe Rad17 amino acid sequence. Further searches of the public data bases using the clone 515944-derived DNA sequence as the query sequence identified several expressed sequence tags with significant sequence similarity. One of these (accession number AA287094) extended the putative Hrad17 sequence in a 5Ј direction and maintained the similarity to S. pombe rad17. This sequence did not extend to the 5Ј end of the ORF, and comparison of the derived amino acid sequence with the S. pombe rad17 ϩ sequence suggested that approximately 60 nucleotides were missing. To complete the putative Hrad17 ORF, a 5Ј RACE-PCR was carried out using a Marathon-Ready human placental cDNA library (CLONTECH) and two gene-specific primers. Approximately 350 nucleotides of novel 5Ј DNA sequence were obtained, including 57 nucleotides required to complete the Hrad17 ORF and 300 nucleotides of 5Ј-untranslated region. Termination codons were present in all three reading frames in the 80 nucleotides immediately 5Ј to the putative Hrad17 initiation codon, indicating that we had isolated the complete coding region (Fig. 1). The full-length Hrad17 ORF was then amplified from cDNA prepared from human SK-N-MC neuroblastoma cells, and the insert DNA sequence from three independent clones was determined. The complete coding region of Hrad17 is 2010 nucleotides in length and has 49% identity to the S. pombe rad17 ϩ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level (Fig. 2). The Hrad17 sequence has been deposited into GenBank TM under accession number AJ004977. Hrad17 also has significant sequence similarity to S. cerevisiae RAD24 (Fig. 2). The Hrad17 cDNA encodes a protein with a predicted molecular mass of approximately 71 kDa.
Northern Blot Analysis for Hrad17-The transcript profile of Hrad17 was examined by probing two multiple tissue Northern blots (CLONTECH) and a cancer cell line Northern blot (CLONTECH) with a probe corresponding to the complete Hrad17 ORF. A single transcript of approximately 3.0 kb was identified for Hrad17. The blots were also hybridized with a ␤-actin probe to demonstrate equal loading of RNA across all lanes. The Hrad17 transcript was present in all tissues examined and was highly elevated in testis. Although the ␤-actin signal is slightly elevated in the cancer cell lines when compared with the normal tissue blots, it is clear that the level of Hrad17 transcript is dramatically elevated in the cancer cell lines examined (Fig. 3). Because the Hrad17 sequence that we have identified is over 2800 bp long, it is likely to include most of the 5Ј-and 3Ј-untranslated region, considering that a typical mammalian poly(A) tail can be several hundred nucleotides long. The presence of an oligo(dT) stretch at the 3Ј end of the clone supports the conclusion that we have the full 3Ј-untranslated region.
Hrad17 Does Not Complement the S. pombe rad17 Checkpoint Phenotypes-Complementation has often been used to demonstrate biological activity for mammalian homologues of yeast proteins (9,28). We examined whether Hrad17 could complement the UV irradiation (DNA damage-dependent checkpoint) and hydroxyurea (DNA replication-dependent checkpoint) sensitivity phenotypes of an S. pombe rad17::ura4 ϩ strain.
Hrad17 was cloned into the S. pombe expression vector pREP3x (22) and transformed into wild type and rad17::ura4 ϩ cells. Transformants were exposed to varying doses of UV or transiently exposed to 10 mM hydroxyurea as described previously (15). We observed no complementation of the UV or hydroxyurea sensitivity phenotypes (data not shown).
Hrad17 Is Located on Chromosome 4q13.3-21.2-The chromosomal position of Hrad17 was determined to establish whether loss of heterozygosity associated with Hrad17 might be linked with any known disease. The 2.6-kb cDNA corresponding to expressed sequence tag clone number AA287094 was used as a probe for FISH mapping. Under the conditions used, the hybridization efficiency was approximately 71% for the probe (among 100 checked mitotic figures, 71 showed signals on one pair of chromosomes). No additional locus was picked up by FISH detection under the conditions used. The 4Ј,6-diamidino-2-phenylindole (DAPI) banding pattern was used to establish that Hrad17 localizes to the long arm of chromosome 4 (25). The detailed position was further determined based upon the analysis of 10 photographs leading to the conclusion that Hrad17 is located on human chromosome 4q13.3-q21.2 (Fig. 4). Loss of heterozygosity of this region of chromosome 4 has been linked to a variety of human neoplasias including breast cancer, hepatocellular carcinoma, and small cell lung cancer (29 -34).
Hrad17 Interacts with Hrad1-The yeast two-hybrid system has been used extensively to examine protein-protein interactions (35)(36)(37). We cloned Hrad17, Hrad9, and two forms of Hrad1 generated by alternative splicing (49), into the yeast two-hybrid expression vectors pAS2-1 and pACT (CLONTECH) and then transformed them individually or as pair-wise combinations into S. cerevisiae strain Y187 (CLONTECH). Transformants were assayed for ␤-galactosidase activity either on filter lifts or using O-nitrophenyl ␤-D-galactopyranoside as substrate as described by the manufacturer (CLONTECH). The ␤-galactosidase activity was high in cells transformed with Hrad17 combined with either form of Hrad1, indicating a positive interaction (Fig. 5). In addition, Hrad17 was able to interact with itself to a lesser extent. Hrad9 did not show any interaction with itself nor with Hrad1 or Hrad17 (Fig. 5). The interaction between Hrad17 and Hrad1 was only observed when Hrad1 was fused with the GAL4 activation domain. We interpret this to mean that the 163-amino acid GAL4 DNAbinding domain fused to the N terminus of Hrad1 was hindering the interaction with Hrad17.

DISCUSSION
We have identified a human cDNA encoding a protein that shares significant sequence similarity with the S. pombe Rad17 protein and the S. cerevisiae RAD24 protein. Alignment of these sequences identifies several blocks of sequence similarity that are conserved between the human and yeast proteins and the human replication factor C subunits (15). One of these contains the consensus sequence for a nucleotide-binding domain. This domain is required for Rad17 function in S. pombe (15). The other regions of sequence similarity are not reminiscent of any defined functional domain but may reflect shared biological activity such as association with elements of the replication machinery or binding of specific DNA structures (15).
The Hrad17 transcript is present in all tissues that we examined and increased in all the cancer cell lines examined, suggesting either that transcription of Hrad17 is proliferationdependent or that it is increased in response to the genomic instability of cancer cell lines. The level of Hrad17 transcript in testis is approximately 10-fold that seen in other tissues. Several yeast cell cycle checkpoint genes play important roles in meiosis (38), and recently ATM and ATR (the human homologue of S. pombe Rad3) were shown to be highly expressed in testis where they interact with meiotic chromosomes. This suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination (39). Hrad1 is also highly expressed in testis (49), suggesting that Hrad1 and Hrad17 may form part of a recognition complex in association with ATM or ATR.
Loss of checkpoint function has been shown to lead to genomic instability even in the absence of exogenous DNA damage (40). In man the p53 gene and the ATM gene are required for the G 1 -S phase checkpoint (41,42). These genes also act as tumor suppressors (43)(44)(45)(46), suggesting that it is likely that other checkpoint genes will act as tumor suppressors. We have shown that Hrad17 resides on chromosome 4 position q13.3-21.2. Loss of heterozygosity of this region of chromosome 4 has been linked to a variety of human neoplasias including breast cancer (29), hepatocellular carcinoma (30), and small cell lung cancer (13,32). Consequently, Hrad17 should be considered as a candidate tumor suppressor gene on chromosome 4q. There is also evidence for the presence of an as yet unidentified gene on chromosome 4q that regulates DNA replication in response to DNA damage (33) and that is involved in the G 1 arrest in cellular senescence (34). Hrad17 should also be considered as a potential candidate for these gene functions.
Demonstration of an interaction between Hrad17 and Hrad1 provides further evidence that we have identified the human homologue of S. pombe rad17. In fission yeast these two gene products act early in both the DNA damage-dependent and DNA replication-dependent checkpoint pathways, and mutants in either gene have similar phenotypes. In the budding yeast, RAD24 and RAD17 (the homologue of S. pombe rad1) are only required for the DNA damage-dependent checkpoint and have been shown to function in conjunction with MEC1 to activate DNA degradation (47), leading to the suggestion that there is a requirement to process single-or double-stranded breaks such that single-stranded DNA is exposed to activate the checkpoint (47,48). Two forms of Hrad1 have been identified; Hrad1A which has an exonuclease activity and Hrad1B, an inactive N-terminal truncation of Hrad1A (49). We show in our twohybrid assay that Hrad17 interacts with both forms of Hrad1. We also find that Hrad17 is able to interact with itself suggest- ing the presence of a multi-subunit complex consisting of Hrad1 and one or more Hrad17 molecules. We were unable to demonstrate the interaction between Hrad17 and Hrad1 when Hrad1 was expressed as a GAL4 DNA-binding domain fusion. Given the small size of Hrad1 we interpret these results to indicate that the fusion is inhibiting the interaction with Hrad17. In the absence of more sophisticated biochemical reagents such as antibodies to Hrad1 and Hrad17, we are unable to confirm the interaction between Hrad1 and Hrad17 by an independent method. Given the sequence similarity between Hrad17 and replication factor C, it is possible that Hrad17 is a DNA-binding protein or interacts with DNA replication proteins. Hrad17 might then act to bring other components of the checkpoint complex such as Hrad1 into close proximity with DNA. The interaction with Hrad1 would provide the exonuclease activity required for the degradation of DNA as proposed by Lydall and Weinert (47).
Hrad17 may also preferentially recognize damaged DNA; however, to assess any potential DNA binding activity we will need to purify Hrad17. Our attempts to express and purify Hrad17 overexpressed in Escherichia coli have been unsuccessful. We find that Hrad17 is extremely unstable when overexpressed, a situation that has also been observed for the S. pombe Rad17 protein.
In summary, these results describe a human homologue of S. pombe Rad17 that interacts with Hrad1 but not Hrad9. This provides further evidence of the conservation between yeast and mammals of checkpoint pathways at the molecular level. A detailed analysis of the interactions between Hrad17 and other checkpoint and DNA replication proteins and a biochemical analysis of the properties of Hrad17 are now required to fully establish the role that this protein plays in cell cycle checkpoint control.