Double‐stranded breaks in S. cerevisiae: recruitment of TFIIH and stability of DNA ends

Abstract

Transcription factor IIH (TFIIH), with known functions in the nucleotide excision repair pathway (NER) and transcription initiation, has been implicated as having a role in the repair of Double Strand Breaks (DSBs) in S. cerevisiae. The Bailis research group identified the Rad3 helicase, a subunit of TFIIH, as an important factor in suppressing short sequence recombination (SSR). The RAD3 mutant, rad3‐G595R, has been shown to increase the frequency of SSR by increasing the stability of DSB ends. In this study we set to determine if TFIIH is physically present at DSBs such that it could be directly affecting DNA end processing. Chromatin immunoprecipitation (ChIP) assays were used to study the recruitment of TFIIH to the site of DSBs in both wildtype and rad3‐G595R mutant strains. Our results indicate that in wild type strains the level of TFIIH at the site of the break increases after a DSB has been induced and then gradually declines once the break stops being induced. However, in a rad3‐G595R mutant there is no significant increase in TFIIH localization following DSB induction. By verifying the presence of TFIIH at DSBs, a role for this complex in dealing with the repair of DSBs has been confirmed. The faulty recruitment observed in the mutant strain helps postulate new ideas on the exact role TFIIH may be playing in DSB repair.(Funding: Pomona College Summer Undergraduate Research Program)