|Home | About | Journals | Submit | Contact Us | Français|
Homologous recombination is an important mechanism for the repair of damaged chromosomes, for preventing the demise of damaged replication forks, and for several other aspects of chromosome metabolism and maintenance. The homologous recombination reaction is mediated by the Rad51 recombinase. In the presence of ATP, Rad51 polymerizes on single-stranded DNA (ssDNA) to form a nucleoprotein filament that is commonly referred to as the “presynaptic filament.” The presynaptic filament is capable of locating a homologous duplex DNA molecule and catalyzing invasion of the duplex to form a DNA displacement loop called the “D-loop.” This protocol describes an in vitro D-loop assay that uses a radiolabeled ssDNA oligonucleotide and a nonlabeled homologous supercoiled duplex DNA as substrates, and agarose gel electrophoresis together with PhosphorImaging for product analysis. To enhance the efficiency of the D-loop reaction, an ancillary factor (the Hop2-Mnd1 complex or Rad54) is included in the reaction. This reconstituted system provides researchers a biochemical means to dissect the mechanisms of the homologous recombination machinery.
It is imperative that highly purified proteins are used to avoid artifacts arising from contaminating nuclease, DNA helicase, or topoisomerase activities. For optimal activity of the purified homologous recombination proteins, avoid repeated freeze-thaw cycles.
|Oligonucleotide D1 (100 μM)||1.5 μL|
|T4 polynucleotide kinase buffer (10X)||10 μL|
|T4 polynucleotide kinase (10 U/μL)||4 μL|
|[γ-32P]ATP, 10 mCi/mL||10 μL|
Problem: D-loop product formation is poor.
Solution: Consider the following:
The studies in the laboratory of the authors have been supported by research grants from the U.S. National Institutes of Health.