Fig. 4: Pol α-primase action at the termini of DNA breaks explains CST-dependent TD formation.

a eGFP sequence with the sgRNA target-sites (in cyan) and PAM-sites (in red). The 6-nitropiperonyloxymethyl (NPOM) modified nucleotides in the right sgRNA are indicated in orange and underlined; theoretical sites of nicking by Cas9-N863A are indicated by triangles (light-induced in orange). b Quantification of the number of tandem duplication (TD) reads in control and CD437 treated mES cells (right) using the experimental timeline shown (left). Data shown represent the mean ± SD, independent experiments are indicated with different symbols. Statistical significance was calculated via a two-tailed paired t-test, *P ≤ 0.05, **P ≤ 0.01. c Graphic representation of the concept of fill-in synthesis at DSBs with 3′ protrusions mediated by primase. The right image illustrates how a stretch of purines, a primase-desert (PD), is hypothesized to prevent the start of fill-in synthesis, hence affecting the length of tandem duplications. d Heat maps representing the relative start position of TDs at 3′ overhangs as illustrated in c for Cas9-N863A-induced DSBs in eGFP (top) and HPRT exon 3 (bottom). e Quantification of the percentage of TDs from (d) that originate from fill-in synthesis starting in the tip (first 15 nucleotides from the 3′ end) of the overhangs of left and right DSB-end. f Heat maps representing the relative start position of TDs at 3′ overhangs for Cas9-N863A-induced DSBs at a random site (top) and at a site containing a PD in the tip of the left overhang (bottom) on chromosome X. g Quantification of the percentage of TDs from (f) that originate from fill-in synthesis starting in the tip (first 15 nucleotides from the 3′ end) of the overhangs of left and right DSB-end. h Heat maps representing the relative start position of TDs on the left and right 3′ overhang of Cas9-N863A-induced DSBs at a site containing a PD in the center of the overhang (top) or at the tip of the left overhang (bottom) on chromosome 8. i Quantification of the percentage of TDs from (h) that originate from fill-in synthesis starting in the tip (first 16 nucleotides from the 3′ end) of the overhangs of left and right DSB-end. All heat maps show three individual experiments per target site in wild-type mES cells and the data is shown as a percentage of the total amount of TDs in the spectra. Data shown in e, g, and i represent the mean ± SEM (n = 3), statistical significance was calculated via a two-tailed unpaired t‐test. ns, not significant, **P ≤ 0.01, ***P ≤ 0.001 ****P ≤ 0.0001. j Model for small tandem duplication formation. We propose that 53BP1-Shieldin-CST mediated fill-in synthesis of DSBs with 3′ overhangs by Pol α-primase leads to suitable substrates for Ku-dependent end-joining, hence resulting in small tandem duplications. Y, pyrimidine; R, purine; PD, Primase Desert; TD, tandem duplication.