The flexible and iterative steps within the NHEJ pathway.

Cellular and biochemical studies of nonhomologous DNA end joining (NHEJ) have long established that nuclease and polymerase action are necessary for the repair of a very large fraction of naturally-arising double-strand breaks (DSBs). This conclusion is derived from NHEJ studies ranging from yeast to humans and all genetically-tractable model organisms. Biochemical models derived from recent real-time and structural studies have yet to incorporate physical space or timing for DNA end processing. In real-time single molecule FRET (smFRET) studies, we analyzed NHEJ synapsis of DNA ends in a defined biochemical system. We described a Flexible Synapsis (FS) state in which the DNA ends were in proximity via only Ku and XRCC4:DNA ligase 4 (X4L4), and in an orientation that would not yet permit ligation until base pairing between one or more nucleotides of microhomology (MH) occurred, thereby allowing an in-line Close Synapsis (CS) state. If no MH was achievable, then XLF was critical for ligation. Neither FS or CS required DNA-PKcs, unless Artemis activation was necessary to permit local resection and subsequent base pairing between the two DNA ends being joined. Here we conjecture on possible 3D configurations for this FS state, which would spatially accommodate the nuclease and polymerase processing steps in an iterative manner. The FS model permits repeated attempts at ligation of at least one strand at the DSB after each round of nuclease or polymerase action. In addition to activation of Artemis, other possible roles for DNA-PKcs are discussed.