Supplementary Figure 2: Ligation of both DNA strands at blunt DNA ends by the NHEJ system and efficiency of ligation by T4 DNA ligase.

(A) (Bottom panel): DNA extension vs. time in the presence of the full complement of NHEJ components. (Middle panel): Supercoiling pattern. (Top panel): Force modulation pattern. The experiment has four phases. (1, light cyan) We calibrate the DNA extension for forces ranging from 1.4 to 0.04 pN. (2, blue) At F~0.4 pN the DNA is at first insensitive to supercoiling as the single covalent bonds joining the bridge and the main segments of the construct act as free swivels. (3, red) Force modulation reveals DNA ligation takes place shortly after injecting the full set of NHEJ components (2000s mark). (4, blue) Now at F~0.4 pN the DNA extension responds to supercoiling indicating both strands of the DNA have been ligated. (B) Time-trace of DNA extension as a function of time upon ligation and cleavage of DNA ends. Force modulation pattern is shown in red. DNA is prepared with 4 bp complementary overhangs using XmaI digest. Addition of T4 DNA ligase (black uparrow) and incubation of DNA at low force rapidly leads to the shortened DNA state which is a hallmark of repair. Addition of SmaI (red uparrow) leads to cleavage of the ligation site and restoration of the DNA extension. (C) Histogram of ∆l values is fit (red line) to a Gaussian distribution with a maximum at 166 ± 10 nm (SD, n = 57 cleavage events). (D) DNA ligation probability per traction cycle for DNA prepared with a 4-base overhang (XmaI digest, blue) or blunt ends (SmaI digest, magenta). In the XmaI-based assay 55/93 DNA molecules underwent ligation. In the SmaI-based assay only 7/84 DNA molecules underwent ligation. Error bars: S.D.