Abstract
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.
Highlights
Cell cycle checkpoints are regulatory pathways ensuring that the events of the cell cycle are completed with high fidelity and in an orderly fashion
Identification of a Human Homologue of S. pombe rad17ϩ— A human expressed sequence tag cDNA clone 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 rad17ϩ 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 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 rad17ϩ sequence suggested that approximately 60 nucleotides were missing
Summary
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. 1–3). Rad17 is not a functional homologue of a replication factor C subunit, 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 rad17ϩ checkpoint gene.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
