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' --> 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.
Highlights
For any dividing cell it is essential to maintain the integrity of the genome ensuring that DNA replication occurs once per cell cycle and that identical chromosomal copies are distributed to two daughter cells at mitosis
In S. pombe, cell cycle checkpoint arrest in response to DNA damage or inhibition of replication is dependent on multiple proteins
Based upon sequence homology and biochemical activity, we have identified a third component, Hrad1, a human homologue of he S. pombe rad1ϩ cell cycle checkpoint gene
Summary
Cloning and Sequencing of Human and Mouse rad1—A search for sequences similar to S. pombe rad1ϩ was carried out using the TBLASTN program [26] against the GenBankTM data base. For the 5Ј RACE, two gene-specific primers were designed to the 5Ј end of the putative Hrad ORF: GSP1 (5Ј-ATTTGTAGGACTTCACTCGTCATAT-3Ј) and GSP2 (5Ј-TGTGTATTGATTTTGCAGACT GTCA3Ј) These were used in a nested PCR reaction with Marathon-Ready human placental cDNA (CLONTECH) as the template. For the 3ЈRACE, two gene-specific primers were designed to the 3Ј end of the putative Hrad ORF: GSP3 (5Ј-CAAGGTTATGGTTACCCTTTGATG-3Ј) and GSP4 (5Ј-TCAATACACAGGAACCTGAGGAG3Ј) These were used in a nested PCR reaction with Marathon-Ready human fetal cDNA (CLONTECH) as the template. The PCR product was digested with BglII and EcoRI and cloned into the corresponding sites of pRSETB (Invitrogen) This creates a fusion protein with the following amino acid sequence N-terminal to the second codon of Hrad1A or Hrad1B: MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSS. FISH signals and the 4Ј,6diamidino-2-phenylindole (DAPI)-banding pattern were recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes [33]
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