Extended Data Fig. 8. SPRTN-dependent proteolysis of the HMCES-DPC is not required for TLS past the AP site.

a, SPRTN immunodepletion. The extracts used in b-f were blotted for SPRTN.

b, A plasmid containing a methylated HpaII DPC that is refractory to degradation by the proteasome (pDPC^me) or pICL^AP were replicated with [α−³²P]dATP in mock- or SPRTN-depleted egg extracts supplemented with p97 inhibitor. Replication intermediates were analyzed as in Fig. 4a.

c, Left, schematic of nascent strands generated during DPC repair. FspI and AatII cut 70 nucleotides to the left and 197 nucleotides to the right of the ICL, respectively, generating characteristic −30 stall, −1 to +1 stall, and strand extension products. Right, pDPC^me was replicated as in b and nascent DNA strands were isolated, digested with FspI and AatII, and resolved by denaturing polyacrylamide gel electrophoresis. As previously reported, SPRTN-depletion delays TLS past the methylated HpaII DPC, as evidenced by the persistence of rightward fork −1, 0, and +1 stall products and a delay in formation of rightward fork extension products.

d, The persistence of the rightward fork −1, 0, and +1 stall products in c was quantified by dividing the summed intensity of the −1, 0, and +1 stall product bands in each lane by the intensity of the full-length rightward extension product band. Quantifications were normalized to the accumulation of the −1, 0, and +1 stall products at the 0 min time timepoint. Quantifications from two independent experiments are shown.

e, Left, schematic of nascent strands generated during AP-ICL repair, as in Fig. 4b. Right, pICL^AP was replicated as in b and nascent DNA strands were analyzed as in Fig. 4b. In contrast to TLS past the methylated HpaII-DPC shown in c, SPRTN-depletion accelerated TLS past the HMCES-DPC, as evidenced by the faster disappearance of rightward leading strand −1 stall products. This result indicates that TLS past the AP site does not require SPRTN-dependent proteolysis of the HMCES-DPC formed during NEIL3-dependent ICL repair.

f, The persistence of the rightward fork −1 stall product in e was quantified as in d. Quantifications from two independent experiments are shown.