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Eukaryotic cells regulate progression through the cell cycle in response to DNA damage. Cell cycle checkpoints are the signal transduction pathways that couple the detection of DNA damage to the proteins that control transitions in the cell cycle. The protein kinase Chk1, originally discovered in fission yeast, but conserved in humans, is essential for preventing mitotic entry in the presence of DNA damage or blocks to DNA replication that cannot be reconciled. Chk1 is phosphorylated in response to DNA damage. Phosphorylation depends on the activity of conserved components of the checkpoint pathway including Rad3, a member of the ATM/ ATR family of kinases. Phosphorylation leads to activation of Chk1 kinase activity. In this chapter, we describe an assay for monitoring the activity of Chk1 isolated. [ABSTRACT FROM AUTHOR]

Genetic screens have been extraordinarily useful for the identification of protein components that function in a variety of cellular processes. For example, a number of proteins that are necessary for responding to deoxyribonucleic acid (DNA) damage were found in screens for loss-of-function mutants that confer hypersensitivity to DNA-damaging agents (1). Genetic screens that report a loss of function can also be used to discern structure-function relationships within a particular protein by providing an assay for loss of function. Combining such an assay with a method that generates mutant alleles of a particular gene can aid in the identification of domains within the encoded protein that are functionally important for the process under investigation. Such approaches are particularly straightforward in unicellular eukaryotes such as Schizosaccharomyces pombe. [ABSTRACT FROM AUTHOR]