The Basic Biology of Aging at the University of Washington
Avraham Shaag

Email:avshaag@u.washington.edu

Dr. Shaag has been working in Mary-Claire Kings lab on the genetic predisposition to old onset breast cancer. Dr. Shaag has focused on Ashkenazi-Jewish families with large numbers of cases of old onset breast and ovarian cancer but with wild type sequence of the BRCA1 or BRCA2 genes. CHEK2, the human homolog of S. cerevisiae Rad53 is a serine/threonine protein kinase (1, 2). CHEK2 functions as a cell cycle checkpoint regulator that is rapidly phosphorylated in response to DNA damage. Activated CHEK2 inhibits CDC25C phosphatase, preventing entry into mitosis, and stabilizes p53, leading to cell cycle arrest. CHEK2 also interacts with BRCA1, allowing BRCA1 to restore survival after DNA damage (3). Using genomic sequencing, Dr. Shaag identified two missense mutations - P85L and S428F - in CHEK2, among the probands of these high-risk families.

In order to evaluate the functional consequences of these variants, Dr. Shaag has developed an assay based on the observation that wildtype human CHEK2 complements deletion of Rad53 in S. cerevisiae (1). Yeast strain Y590 (delRad53::HIS3) and plasmid pMH267(2u LEU2 GAL-CHK2) were kindly provided by Stephen Elledge. By site directed mutagenesis of the pMH267 plasmid, Dr. Shaag cloned mutations S428F, P85L, and 1100delC in the CHEK2 gene. As controls for the mutagenesis procedure, Dr. Shaag also cloned a silent mutation E84E and reverted the S428F mutation to recreate the wildtype sequence, plasmid "F428S. Each mutant plasmid and pMH267 (wildtype human CHEK2) were transformed into the Y590 yeast strain.

As expected, wildtype human CHEK2 complemented the lethality of the Rad53 deletion in yeast, leading to exponential growth in selective media. Yeast carrying human CHEK2 with the P85L variant or the silent mutation E84E also complemented the Rad53 deletion and grew equally well. In contrast, yeast carrying human CHEK2 with the S428F mutation or with the 1100delC mutation grew no better than the Rad53-null yeast with no complementary human gene. As an additional control, the CHEK2.S428F mutation reverted to wildtype ("F428S") complemented Rad53 lethality as well as did wildtype human CHEK2. Results of the functional screen suggested that S428F but not P85L was a pathogenic allele of CHEK2.