Extended Data Fig. 1: NA does not affect replication efficiency or replication fork progression.
From: A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo
Three circular plasmids containing the SV40 origin of replication, and an expanded (CAG)79•(CTG)79 repeat tract (pDM79EF and pDM79HF) or no repeats (pKN16), were replicated in vitro by human (HeLa) cell extracts without or with NA (7.5 µM or 15 µM) treatment. The location of SV40-ori determines the replication direction and which strand will be used as the leading or the lagging strand template. pDM79HF uses the CAG strand as the lagging strand template, while pDM79EF uses the CTG strand as the lagging strand template (schematic on the top of the gel panel). Replication products were purified and linearized with BamHI. An equal portion of the reaction material was also digested with BamHI and DpnI as DpnI digests un-replicated and partially-replicated material, as shown in the schematic (top figure). The digestion products were electrophoresed on a 1% agarose gel to resolve completely replicated and un-replicated material (bottom figure). Equal amount of unreplicated plasmid DNA was digested with DpnI and stained with Ethidium Bromide to show the complete digestion of unreplicated plasmid DNA (Bottom panel). Panel I, ethidium bromide stained, Panel II, autorad: marker (lane 1); DpnI undigested plasmid DNA (lane 2); DpnI digested unreplicated plasmid DNA (lane 3-4); replicated plasmid DNA, DpnI resistant (lane 5). No difference in DpnI resistant material is observed between replication in the presence or absence of NA, in all the three templates tested (panel III, IV, V). Blots have been cropped and the corresponding full blots are available in the Source Data files.
