Journal: bioRxiv

Article Title: Leading and lagging strand abasic sites differentially affect vertebrate replisome progression but involve analogous bypass mechanisms

doi: 10.1101/2025.01.09.632187

Figure Lengend Snippet: A) Independent experimental replicate of . B) Quantification of thetas (θs) from . C) Independent experimental replicate of (B) D) Quantification of catenanes (cats) from . E) Independent experimental replicate of (D) F) Products from at 15 minutes were separated by 2D gel electrophoresis to visualize sigmas (σs), replication intermediates and catenanes (RIs+Cats), nicked Circular Monomers (nCMs), and gapped Circular Monomers (gCMs). G) Quantification of σs from (F). H) Independent experimental replicate of (G). I) To assess the presence of DNA-protein cross-links (DPCs), replication products were generated as in , treated with or without Proteinase K, then separated on a native agarose gel and visualized by autoradiography (as in ). Replication of control DNA in the presence of etoposide (100 μM, E) was used as a positive control for DPC formation. Etoposide treatment resulted in a noticeable decrease in mobility when Proteinase K treatment was omitted (compare lanes 1 and 14) due to DPC formation, as expected . In contrast, omission of Proteinase K had little effect on DNA structures arising from replication of pCTRL or pAP (compare lanes 2-13 and 15-26). Thus, there was no evidence of DPC formation during replication of pAP. J) Independent experimental replicate of . K) Quantification of supercoiled circular monomers (scCMs) as a percentage of total circular monomers (including nicked and gapped circular monomers (nCMs and gCMs)) from to estimate frequency of stalling at AP sites. Replication of a strand-specific DNA lesion in Xenopus egg extracts is expected to result in one fully replicated daughter molecule (scCM) and one nicked or gapped daughter molecule (nCM or gCM) . Thus, if all AP sites are retained and stall DNA synthesis, half the daughter molecules are expected to be replicated with the same timing as undamaged plasmid DNA (pCtrl). To estimate timing of replication for a single undamaged DNA strand, half of the signal from the control condition was plotted (pCtrl (50%)). At 10- and 15-minutes signal from replication of pAP closely matched signal from pCtrl (50%), consistent with half of DNA strands being replicated with normal timing in the pAP condition. The datapoints then diverged once pCtrl (50%) plateaued, consistent with half of DNA strands being replicated much more slowly in the pAP condition. Thus, these data suggest that most AP sites are retained and stall replication on one strand only.

Article Snippet: Where indicated, NPE alone was supplemented with etoposide (Etop; Sigma) to a final concentration of 100 μM in the reaction, and ubiquitin vinyl sulfone (UbVS; R&D Systems) was added to both the licensing mix and 50% NPE (v/v%) at a final concentration of 20 μM as previously described ( ).

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Generated, Agarose Gel Electrophoresis, Autoradiography, Control, Positive Control, DNA Synthesis, Plasmid Preparation