Protocol

DNA Repair Protocols

Volume 920 of the series Methods in Molecular Biology pp 349-370

Date:

Monitoring DNA Recombination Initiated by HO Endonuclease

  • Neal SugawaraAffiliated withDepartment of Biology, Brandeis UniversityRosenstiel Basic Medical Sciences Research Center, Brandeis University
  • , James E. HaberAffiliated withDepartment of Biology, Brandeis UniversityRosenstiel Basic Medical Sciences Research Center, Brandeis University Email author 

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Abstract

DNA double-strand breaks (DSBs) have proven to be very potent initiators of recombination in yeast and other organisms. A single, site-specific DSB initiates homologous DNA repair events such as gene conversion, break-induced replication, and single-strand annealing, as well as nonhomologous end joining, microhomology-mediated end joining, and new telomere addition. When repair is either delayed or prevented, a single DSB can trigger checkpoint-mediated cell cycle arrest. In budding yeast, expressing the HO endonuclease under the control of a galactose-inducible promoter has been instrumental in the study of these processes by providing us a way to synchronously induce a DSB at a unique site in vivo. We describe how the HO endonuclease has been used to study the recombination events in mating-type (MAT) switching. Southern blots provide an overview of the process by allowing one to examine the formation of the DSB, DNA degradation at the break, and formation of the product. Denaturing gels and slot blots as well as PCR have provided important tools to follow the progression of resection in wild-type and mutant cells. PCR has also been important in allowing us to follow the kinetics of certain recombination intermediates such as the initiation of repair DNA synthesis or the removal of nonhomologous Y sequences during MAT switching. Finally chromatin immunoprecipitation has been used to follow the recruitment of key proteins to the DSB and in subsequent steps in DSB repair.

Key words

HO Meganuclease Recombination Double-strand break Gene conversion Mating-type switch Chromatin immunoprecipitation