Alternative DNA Damage Checkpoint Pathways in Eukaryotes
Abstract
The human CHES1 (checkpoint suppressor 1) gene is sufficient to restore DNA damage-induced G2 arrest in multiple S. cerevisiae checkpoint mutants by activation of an alterative checkpoint pathway. Our goal is to identify the CHB (checkpoint bypass pathway) genes that constitute this alterative checkpoint, to isolate the human counterparts of these genes, and to compare their structure and activity in normal and cancer tissues. In an effort to identify the genes involved in the alternative pathway, we performed a comprehensive screen in S. cerevisiae by mutagenizing a cdc9-8, rad9delta mutant strain, which is both UV-resistant and grows at 30C in the presence of CHES1. The screen resulted in three mutants that display varying degrees of temperature- and UV-sensitivity. In summary, chb13 is a strong mutant in which CHES1 can no longer suppress either the temperature or UV-sensitive phenotypes chb16 and chb57 are highly temperature- sensitive/ moderately UV-sensitive and highly UV-sensitive/moderately temperature-sensitive, respectively. Despite multiple attempts, we were unable to identify the gene(s) mutated in the chb13 mutant strains. We have taken an alterative approach to this goal by using an S. cerevisiae insertional mutagenesis method, which will avoid the cloning problems experienced in using the previous EMS methodology. In addition, in efforts to identify CHES1 1-interacting proteins by biochemical methods, we have constructed a human SOS (son of sevenless)-CHESl bait construct and a GST (Glutathione-S-Transferase)-CHES1 fusion construct for use in a cytoplasmic yeast-two-hybrid screen and GST pulldown experiments, respectively. The insertional mutagenesis screen, the two-hybrid screen, and the pulldown experiments are currently in progress.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 01, 2001
- Accession Number
- ADA396817
Entities
People
- Kenneth L. Scott
- Sharon E. Plon
Organizations
- Baylor College of Medicine