Systems and methods for single-strand break signaling and repair in a cell-free system and methods of identifying modulators of single-strand break signaling and repair

A method for identifying modulators of single-strand break (SSB) DNA damage response (DDR) activity, the method comprising:

• providing a composition comprising a plurality of engineered, site-specific, nicked, SSB plasmid structures, each having an inner (−) and outer (+) strand and genetically modified to comprise a single nick in the +strand only, the nick located at a single recognition site for a specific restriction enzyme and modified to have a hydroxyl group at each nicked end of the SSB plasmid structure;
• providing a replication-independent, eukaryotic, cell-free extract capable of replication-independent initiation of one or more SSB DNA damage response (DDR) activities when incubated with the composition of engineered site-specific, nicked, SSB plasmid structures;
• combining the composition of engineered, site-specific, nicked, SSB plasmid structures with the eukaryotic cell-free extract and a test compound to make a test mixture;
• and
• detecting a SSB DDR activity.