Suicide deletion (Figure 1) is not a research project per se, but rather is a very powerful research tool we developed in our early years. The real benefits of suicide deletion are its simplicity, speed, ready use in genetic screens, adaptability to testing different DSB configurations, and statistical reliability. We are currently exploiting these features in a series of new experimental frontiers.
In the suicide deletion assay, an endonuclease (typically I-SceI) is expressed in yeast that cuts at a specific site in a chromosome, thereby creating a DSB. The unique aspect of our approach is that we in fact cut at two I-SceI recognition sites so that the resulting DSB has excised the coding sequence of the I-SceI gene and thereby terminates endonuclease expression. This allows us to monitor the simple rejoining of I-SceI cut sites by NHEJ, which is otherwise impossible since the repaired site would simply be recleaved. Click here for more details.
Figure 1. The suicide deletion assay of DSB repair.
As an example, an initial application of suicide deletion was to screen all viable yeast gene deletion mutants for a specific defect in NHEJ (Figure 2). This analysis revealed all known NHEJ genes (of the Ku, DNA ligase IV and Rad50-Mre11 complexes), as well as the previously unknown NHEJ gene NEJ1. In addition, because of the comprehensive nature of this genetic screen, we could be (reasonably) confident that no other viable mutants will show a strong NHEJ defect. Click here for more details.
Figure 2. A near-comprehensive screen of yeast mutants for NHEJ defects.
