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

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 Hrad1 alternative 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 that Hrad1 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 cerevisiae RAD17 and Ustilago maydis REC1 proteins. REC1 has previously been characterized as a 3 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 3 5 exonuclease activity, whereas HisHrad1B lacks such activity. The biological functions of the two proteins remain to be determined.

Human Homologs of Schizosaccharomyces pombe Rad1, Hus1, and Rad9 Form a DNA Damage-responsive Protein Complex

“…hRad1, hRad9, and hHus1 Associate in a Checkpoint Complex-Comparisons of the predicted human and yeast protein sequences indicate that the human proteins are 25-30% identical and 53-57% similar to their respective homologs, with homologies extending over extensive portions of each protein (12)(13)(14)(15)(16)(17)(18). In S. pombe, spRad1 and spHus1 associate in wildtype, but not rad9, mutant yeast.…”

“…The similarities between yeast and humans extend beyond these proteins, as human homologs of sprad1 (12)(13)(14), sprad9 (15), sprad17 (16), spchk1 (17), and sphus1 (18) genes have been identified, fueling speculation that the checkpoint pathways may be conserved. Despite these advances, the physical and functional roles of these proteins are largely unexplored, even in the well studied yeast systems.…”

DNA damage checkpoint control in cells exposed to ionizing radiation

“…The 9-1-1 complex is composed of the RAD9, HUS1 and RAD1 subunits that interact to form a PCNA-like structure that loads on the DNA like a sliding clamp similar to PCNA. It has also been speculated that the 9-1-1 complex functions to generate more ssDNA to enhance checkpoint-associated signaling (Lydall and Weinert, 1995;Parker et al, 1998;Volkmer and Karnitz, 1999;Xu et al, 2001). It will be interesting to see whether and how the RAD16-RFC and 9-1-1 complexes are involved in checkpoint signaling originating from sites of DNA DSBs.…”

Colocalization of human Rad17 and PCNA in late S phase of the cell cycle upon replication block

“…By yeast two hybrid analysis it was shown that hRad17 interacts with hRad1 (Parker et al, 1998). The checkpoint proteins hRad9, hHus1, and hRad1 can be co-immunoprecipitated with hRad17 from human cell extracts (Rauen et al, 2000).…”

“…The hRad17/ hRF-C complex is thought to load the 9-1-1 complex onto the DNA at the site of a DNA lesion during an early stage of checkpoint activation (Mitchell, 2001). Recombinant hRad1 and hRad9 purified from E. coli exibit 3'-5'exonuclease activity (Parker et al, 1998;Bessho and Sancar, 2000), suggesting that these proteins might play a role in the processing of DNA lesions.…”