Defects in a cell's ability to maintain its genomic integrity upon DNA damage and replication stress can lead to cancer. Recent evidence suggests that in addition to DNA repair enzymes, recruitment of enzymes that modify the chromatin landscape surrounding the site of damage is necessary for efficient repair. INO80 is an ATP dependent chromatin remodeling enzyme from S. cerevisiae. INO80 is recruited to sites of genomic instability, such as double strand breaks and stalled replication forks, where its enzymatic activity is required for normal kinetics of double strand break repair and replication re start. Specifically, INO80 is required for histone eviction near the break and thus recruitment of key DNA repair factors. However, how INO80 directly affects the structure of nucleosomes surrounding damaged DNA is unknown. In order to address this question, we propose to take an enzymological approach using purified enzyme and reconstituted nucleosomes to quantitatively characterize the mechanism of INO80. We hypothesize that the enzymatic activity of INO80 is directly regulated by damaged DNA through the intrinsic specificity INO80 has for nucleosomes that signal damaged DNA. To test this hypothesis, we will use a combination of FRET based, gel based, and restriction enzyme based methods to quantify the kinetics of both fast steps and slow steps of nucleosome remodeling and test how the steps of remodeling are changed in the presence of nucleosome substrates that specific to damaged DNA.
In Aim 1, we will investigate the effect that broken DNA ends or single stranded overhangs have on the remodeling and ATPase activity of INO80, and in Aim 2, we will investigate the effect of damage- specific histone modifications on the same activities. Understanding how INO80 responds to these damage- specific nucleosomes will further our understanding of how the chromatin landscape must be actively altered under conditions of genomic instability in order to facilitate repair of damaged DNA. This knowledge will useful in cancer therapies that target chromatin remodeling specifically under conditions of genomic instability.
DNA damage during replication leads to genomic rearrangements and mutations that may cause cancer. INO80 is an ATP dependent chromatin remodeling enzyme that plays a key role in DNA repair. INO80 changes the structure of the chromatin surrounding DNA damage to create access for DNA repair proteins. However, the mechanism of INO80's enzymatic activity at sites of DNA damage is unknown. The goal of our research is to determine how the enzymatic activity of INO80 is modulated by chromatin substrates that signal DNA damage. The results of this research will help us better understand how chromatin must be altered in order to facilitate DNA repair. We expect that the results of this study will contribute to our ability to target chromatin remodeling enzymes for cancer therapeutics.