Fig. 6: 53BP1 promotes TLS in human cell extracts.

a Sequence detail of the lesion-containing shuttle plasmid used in in vitro DNA replication reactions in the presence of SV40 T antigen, indicating the staggered position of TT(CPD) lesions on each strand. Below, typical sequence outcomes detected in PCR products of DpnI-digested replicated DNA are shown (top strand sequence), together with the responsible mechanisms. CPD, cyclobutyl pyrimidine dimer, TLS, translesion synthesis, NER, nucleotide excision repair, ssDNA, single-stranded DNA. b Western blot of 53BP1 in samples of cytosolic and nuclear extract from WT or 53BP1^–/– human TK6 cells as used in subsequent experiments. A representative image of three experiments is shown. c The efficiency of replication, as measured by the ratio of quantitative PCR products spanning 3 or 0 DpnI sites on the lesion-containing plasmids recovered from 10 μl reactions that contained the indicated amounts of cytosolic (cyt) or nuclear extracts (nx) from WT or 53BP1^–/– TK6 cells and the indicated concentrations of a recombinant 53BP1 fragment. d Sequence outcomes in the replication reactions also shown in panel (c), measured using sequence-specific quantitative PCR on longer PCR products derived from DpnI-resistant replicated lesion-containing plasmids which were incubated in the indicated extracts. TSw, template switching. Means and individual values of independent experiments are shown in (c, d), the significance of tested differences is indicated (ns not significant, unpaired two-sided t-test, n = 3 independent experiments except n = 2 for the reconstitution experiments). e A schematic model of the choice between DNA damage bypass mechanisms that influence mutagenesis in the presence or absence of BRCA1/2. Yellow symbols represent RAD51. Source data are provided as a Source Data file.