Dissecting BRCA1-PALB2 Activity in DNA Repair and Development

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PROJECT SUMMARY/ABSTRACT This submission is for a supplement award for the parent grant R01GM135293: “Dissecting BRCA1-PALB2 activity in DNA repair and development”. The parent grant is currently in its first year, making this the perfect time to upgrade our equipment. The localization of proteins to double stranded DNA breaks (DSBs) can be quantified by the presence of nuclear foci. The number of foci within a nucleus is often indicative of whether a particular protein or repair pathway is functional. A typical focus appears as a homogenous sphere under conventional widefield or confocal microscopy. The information provided does not go beyond whether foci are present or absent. In contrast, super-resolution (SR) microscopy provides high-resolution imagining, which can reveal detailed insights into the DSB-flanking chromatin topology. The ability to perform SR microscopy would greatly enhance the knowledge that can be gained from the current R01-funded project. Not only in understanding the effects of mutations on foci formation, new insights into the mechanics of the DNA damage repair machinery and the interplay between chromatin topology and DNA repair can be revealed. In the parent grant, we aim to uncover the mechanisms by which BRCA1 recruits PALB2 to sites of DNA damage. We proposed to examine the effects of BRCA1 and PALB2 mutations on DNA repair foci. However, SR microscopy has the potential to reveal so much more, beyond simply the presence or absence of foci, such as insights into the nanostructure of BRCA1-PALB2, whether there are separate or overlapping nanodomains, spatial differences, and the BRCA1-PALB2 topology relative to additional homologous recombination (HR) proteins. Moreover, the effects of BRCA1 and PALB2 patient mutations have previously not been examined for their effects on DSB-chromatin topology and nanostructure. The use of SR microscopy will provide super-resolution insight, and ultimately improve our understanding of how BRCA1 and PALB2 mutations impact human health.