The essential elements for the noncovalent association of two DNA ends during NHEJ synapsis

One of the most central questions about the repair of a double-strand DNA break (DSB) concerns how the two free DNA ends are brought together — a step called synapsis. Using single-molecule FRET (smFRET), we show here that both Ku plus XRCC4:DNA ligase IV are necessary and sufficient to achieve a flexible synapsis of blunt DNA ends, whereas either alone is not. Addition of XLF causes a transition to a close synaptic state, and maximum efficiency of close synapsis is achieved within 20 min. The promotion of close synapsis by XLF indicates a role that is independent of a filament structure, with action focused at the very ends of each duplex. DNA-PKcs is not required for the formation of either the flexible or close synaptic states. This model explains in biochemical terms the evolutionarily central synaptic role of Ku, X4L4, and XLF in NHEJ for all eukaryotes.

glucose plus gloxy oxygen scavenger system. Data is represented as mean ± SD of six replicates. c, Histogram and corresponding exponential fit of total synapsis time mediated by Ku and X4L4 in the glucose plus gloxy oxygen scavenger system. Cy3 signal lifetime (including zero-FRET and detectable FRET portions) of the synaptic complex was used to calculate the total dwell time for each synapsis event and only the synapsis events (n = 277) with both start and end time points within the detection time window were included. Synapsis time shown on the graph was represented as mean ± S.D. of three independent repeats. d, Normalized synapsis efficiency. Data are represented as mean ± SD of at least two independent replicates. The data shown on lane 1 and lane 2 are the same as shown on Fig. 2a. The t-test (unpaired, two-tailed) was applied for p value calculation. e,f, One of the dynamic intensity traces (donor: green; acceptor: magenta) and corresponding E FRET values (blue) of synapsis mediated by Ku and X4L4 (d) or by Ku, X4L4, and DNA-PKcs (e) in the glucose plus gloxy oxygen scavernger system . The black line represents the smoothed trace of corresponding donor, acceptor, or E FRET trace. g, Autocorrelation curves for the dynamic synaptic complexes and corresponding bi-exponential fit. The synaptic complex was formed by Ku and X4L4, and by Ku, X4L4, and DNA-PKcs, and the study used the glucose plus gloxy oxygen scavenger system in the solution. The t-test (unpaired, two-tailed) was applied for p value calculation. Source data are provided as a Source Data1 file.

Cy5
BZ15/HC120 74bp Cy3   TAG TGG GTT CAG CAG GCA TTG TGC TAT  GAT CAA CCG AAT CTG TAC ATA TAT CAG TGT CTG  have a kb of naked DNA exposed.

5'PO
Here we found the PCA plus PCD oxygen scavenger system severely reduces the kinase activity of DNA-PKcs. The same PCA plus PCD system was used in the study by Graham et al 1 . Though they proposed that DNA-PKcs kinase activity is required for transition from one synaptic state to another, this conclusion must be re-evaluated because the DNA-PKcs kinase was almost certainly inactive due to the use of PCA plus PCD.
Given the requirement of DNA-PKcs for activation of the endonuclease activity of Artemis at DNA ends 2 , we speculate that instead of being required for blunt end synapsis, DNA-PKcs plus Artemis may be important for processing incompatible DNA ends prior to synapsis. Although DNA-PKcs does not show any significant effect on end synapsis in our in vitro highly purified system here, there is always the possibility that a protein not included 13 could reveal such a role for DNA-PKcs or that DNA-PKcs in vivo is aided by other proteins for end synapsis. Future work is required to address this possibility.
The flexibility and the physical gap between the two dsDNA ends within the FS complex may facilitate the end processing by other NHEJ factors, such as nucleases and polymerases [3][4][5]  The 5' OH at the dsDNA ends precludes ligation and thus permits us to focus only on the synapsis process. In addition to its requirement for covalent ligation, the 5' PO4 at each end could well have some effects on synapsis. Further work will be required to examine this issue.

PAXX Can also Drive Two dsDNA Ends into Close Proximity.
PAXX is known to promote NHEJ in cells [9][10][11] . Our synapsis results here show that PAXX indeed can drive the dsDNA ends into a close synapsis (CS). That PAXX can independently function like XLF to promote CS but at a lower efficiency is in agreement with previous findings that PAXX is dispensable for normal mouse development [12][13][14] and V(D)J recombination in vivo 15,16 . It is also in agreement with in vitro biochemical studies showing that PAXX and XLF have overlapping functions in ligation 16 . The consistent nature of our smFRET with these in vivo results suggest that it is the capability for synapsis that limits the NHEJ activities of different factors. This emphasizes the importance and rate-limiting nature of the synapsis step.
14 PAXX can interact with Ku70 through its C-terminal end, but with a lower affinity compared to XLF binding to Ku 16 , which may explain the much lower efficiency of CS complex formation promoted by PAXX. The overlapping function of PAXX and XLF, and the lower CS formation efficiency of PAXX suggest that the role of PAXX in synapsis is masked by XLF, as observed in ligation 16 .

Synapsis Stimulated by XLF is Primarily End-to-End.
Previous structural investigations and single molecule studies suggested that X4-XLF or X4-XLF-L4 can form filaments, which could be important for these proteins to mediate synapsis 8,[17][18][19][20] . Therefore, the filament model was proposed to mediate synapsis at the earliest stages of NHEJ. XLF promotes the formation of the CS complex, in which the two dsDNA molecules make contact at the ends, which indicates that XLF facilitates end-to-end synapsis. The result that XLF can stimulate the formation of end-to-end CS complex, suggests a non-filament role for XLF in promoting synapsis, given that a filament interaction primarily occurs in the mid-section of the dsDNA. Though synapsis could be detected using the duplex with the FRET acceptor labeled at a recessed (mid-point) position in the duplex, the number of detected non-zero FRET molecules is much lower than that found when the acceptor is at the end. This further illustrates that synapsis stimulated by XLF is primarily end-to-end. Importantly, though not likely at play in our purified system, it is quite possible that filaments of XLF could be relevant for chromatinized DNA. Eventual high-resolution cellular work and utilization of chromatinized DNA substrates in biochemical systems will be valuable in this regard.