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Replication forks (RFs) frequently encounter barriers or lesions in template DNA that can cause them to stall and/or break. Efficient genome duplication therefore depends on multiple mechanisms that variously act to stabilize, repair, and restart perturbed RFs. Integral to at least some of these mechanisms are homologous recombination (HR) proteins, but our knowledge of how they act to ensure high-fidelity genome replication remains incomplete. To help better understand the relationship between DNA replication and HR, fission yeast strains have been engineered to contain intrachromosmal recombination substrates consisting of non-tandem direct repeats of ade6 heteroalleles. The substrates have been modified to include site-specific RF barriers within the duplication. Importantly, direct repeat recombinants appear to arise predominantly during DNA replication via sister chromatid interactions and are induced by factors that perturb RFs. Using simple plating experiments to assay recombinant formation, these strains have proved to be useful tools in monitoring the effects of impeding RFs on HR and its genetic control. The strains are available on request, and here we describe in detail how some of them can be used to determine the effect of your mutation of choice on spontaneous, DNA damage-induced, and replication block-induced recombinant formation.

Original publication

DOI

10.1007/978-1-60327-815-7_31

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2009

Volume

521

Pages

535 - 552

Keywords

DNA Damage, DNA Replication, DNA, Fungal, Genes, Fungal, Mutagens, Mutation, Recombination, Genetic, Repetitive Sequences, Nucleic Acid, Schizosaccharomyces, Ultraviolet Rays