Interplay between nucleosomes, nucleases and cell cycle kinases during DNA end resection

Kurzfassung

DNA double strand breaks (DSBs) can be repaired by two principal cellular mechanisms – recombination-based (such as homologous recombination, HR) and direct ligation-based (such as non-homologous end joining, NHEJ). In mitotically dividing cells HR critically depends on the presence of sister chromatids as repair templates and hence DSB repair pathway choice is highly cell cycle regulated. Regulation occurs primarily at the level of DNA end resection, the first step in the HR reaction. Notably, nucleosomes restrict resection both directly and indirectly by recruiting nucleosome-associated resection inhibitors such as Rad9/53BP1. Nucleosome remodellers – in particular Fun30/SMARCAD1– are recruited to DSBs in order to overcome this inhibition. Our data show how nucleosome remodellers control DNA end resection. Fun30 is under cell cycle control and targeted to DSBs in a cell cycle-regulated manner. Such targeting is efficient long-range resection of DSBs and remarkably, allows to partially bypass the cell cycle regulation of long-range resection, if made constitutive. This suggests that nucleosome remodelling by Fun30 is a critical and conserved mechanism for DSB repair regulation. In order to investigate the fate of nucleosomes during DNA end resection, we established a ChIP-seq-based methodology to measure ssDNA and dsDNA binding of histones in vivo. Our data suggest that nucleosomes are evicted during DNA end resection. A model how nucleosomes may control steps downstream of resection will be discussed. Lastly, we describe a new layer of cell cycle-regulation in the whole process, deregulation of which allows to partially reconstitute DNA end resection and homologous recombination already in the G1 phase of the cell cycle. We will discuss implications for genome editing.