A Human Homologue of the Schizosaccharomyces pombe rad1 + Checkpoint Gene Encodes an Exonuclease

doi:10.1074/jbc.273.29.18332

A Human Homologue of the Schizosaccharomyces pombe rad1⁺ Checkpoint Gene Encodes an Exonuclease*

From the ^‡Department of Experimental Molecular Biology and the ^¶Department of Applied Molecular Biology, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium

Abstract

In the fission yeast Schizosaccharomyces pombe the rad1 ⁺ gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human homologue of the S. pombe rad1 ⁺ gene, designated Hrad1, as well as a mouse homologue: Mrad1. Two Hrad1alternative splice variants with different open reading frames have been identified; one codes for a long form, Hrad1A, and the other encodes a short form because of N-terminal truncation, Hrad1B. Hrad1A has 60% identity to the S. pombe rad1 ⁺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 cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates thatHrad1 is located on chromosome 5p13.2–13.3. This region is subject to loss of heterozygosity in several human cancers. Hrad1 also shares homology with the Saccharomyces cerevisiaeRAD17 and Ustilago maydis REC1 proteins. REC1 has previously been characterized as a 3′ → 5′ exonuclease with a C-terminal domain essential for cell cycle checkpoint function. We have expressed and purified polyhistidine-tagged fusions of Hrad1A and Hrad1B and show that HisHrad1A has 3′ → 5′ exonuclease activity, whereas HisHrad1B lacks such activity. The biological functions of the two proteins remain to be determined.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AJ004974, AJ004975, and AJ004976.
↵§ Present address: Cardiovascular Metabolism and Musculoskeletal Research Dept., Zeneca Pharmaceuticals, Alderley Edge, Cheshire, UK.
↵‖ To whom correspondence should be addressed. Tel.: 32-14-602618 or 32-14-605734; Fax: 32-14-606111; E-mail:wluyten{at}janbe.jnj.com.
↵1 The abbreviations used are: ORF, open reading frame; RACE, rapid amplification of cDNA ends; FISH, fluorescencein situ hybridization; DAPI, 4′,6-diamidino-2-phenylindole; EST, expressed sequence tag; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; kb, kilobase pair(s).
↵2 A. Parker, unpublished results.
- Received December 29, 1997.
- Revision received April 22, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.