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. Author manuscript; available in PMC: 2019 Sep 14.
Published in final edited form as: Science. 2018 Sep 14;361(6407):1126–1129. doi: 10.1126/science.aat2477

Fig. 3: Impact of ribonucleotide termini on the NHEJ ligation step.

Fig. 3:

(A–C) Termini of NHEJ substrates were varied to be consistent with polymerase-dependent addition of a ribonucleotide vs. a deoxynucleotide, and introduced into MEFs expressing neither Pol μ nor TdT. Sensitivity of amplified products to a diagnostic restriction enzyme (RE) was used to identify examples of direct head-to-tail ligation. (B-C) Substrates with indicated terminal nucleotides were introduced into MEFs expressing neither Pol μ nor TdT. The mean % directly ligated products for three independent transfections, ± sd, is noted below. (D) Deoxy-guanine triphosphate (+GTP), ribo-guanine triphosphate (+rGTP), or an equivalent amount of the relevant salt (“none”), were added to Rosa26 Cas9-sgRNP transfections performed as in Fig. 2C–D, and genomic DNA was harvested after 1 hour. Data are the mean from 4 transfections, and error bars represent sd. Means were compared by ANOVA in pairs to no NTP added (***; p < 0.001). (E) Triple strand break repair model. Pol μ or TdT-dependent ribonucleotide addition is noted in red.