Methods for studying mutagenesis and checkpoints in Schizosaccharomyces pombe.
Mutations in genome caretaker genes can induce genomic instability, which are potentially early events in tumorigenesis. Cells have evolved biological processes to cope with the genomic insults. One is a multifaceted response, termed checkpoint, which is a network of signaling pathways to coordinate cell cycle transition with DNA repair, activation of transcriptional programs, and induction of tolerance of the genomic perturbations. When genomic perturbations are beyond repair, checkpoint responses can also induce apoptosis or senescence to eliminate those deleterious damaged cells. Fission yeast, Schizosaccharomyces pombe (S. pombe) has served as a valuable model organism for studies of the checkpoint signaling pathways. In this chapter, we describe methods used to analyze mutagenesis and recombinational repair induced by genomic perturbations, and methods used to detect the checkpoint responses to replication stress and DNA damage in fission yeast cells. In the first section, we present methods used to analyze the mutation rate, mutation spectra, and recombinational repair in fission yeast when replication is perturbed by either genotoxic agents or mutations in genomic caretaker gene such as DNA replication genes. In the second section, we describe methods used to examine checkpoint activation in response to chromosome replication stress and DNA damage. In the final section, we comment on how checkpoint activation regulates mutagenic synthesis by a translesion DNA polymerase in generating a mutator phenotype of small sequence alterations in cells, and how a checkpoint kinase appropriately regulates an endonuclease complex to either prevent or allow deletion of genomic sequences and recombinational repair when fission yeast cells experience genomic perturbation in order to avoid deleterious mutations and maintain cell growth.