Journal: Science Advances

Article Title: Multistep mechanism of G-quadruplex resolution during DNA replication

doi: 10.1126/sciadv.abf8653

Figure Lengend Snippet: ( A ) DHX36-depleted NPE supplemented with buffer, wild-type (WT) DHX36, or DHX36 E327A (EA) were analyzed by Western blot with the DHX36 antibody alongside a dilution series of undepleted extract. ( B ) pG4 Lead was replicated in the indicated extracts and analyzed by ChIP-qPCR with DHX36 (i), MCM7 (ii and iv), or FANCJ (iii) antibodies using a primer pair for the G4 locus (i to iii) or for the lacO locus (iv). Schematic representation of qPCR loci on top. Brown hexamer, CMG; green sphere, LacI. ( C ) pG4 Lead was replicated in the indicated extracts, digested with Afl III, Bam HI, and Cla I (top gel) or Afl III and Bam HI (bottom gel), separated on a denaturing polyacrylamide gel, and visualized by autoradiography. The CMG stalling and polymerase stalling products are indicated with brackets. ( D ) pG4 UP (G4 on bottom strand and T25 upstream of G4) was replicated in the indicated extracts and analyzed as in (C) but only digested with Afl III and Bam HI. Extension product of the nascent leading strand past the G4 is shown in the top panel. Where indicated, LacI was released by addition of isopropyl-β- d -1-thiogalactopyranoside (IPTG) at 15 min (lanes 25 to 48). The CMG stalling and polymerase stalling products are indicated with brackets. To monitor replication fork block by LacI, extracted samples were digested with Hinc II, separated on native agarose gel, and visualized by autoradiography (bottom gels).

Article Snippet: Antibodies used for ChIP experiments were purified with rProtein A Sepharose (PAS) beads (GE Healthcare, catalog no. 171279-01).

Techniques: Western Blot, Autoradiography, Blocking Assay, Agarose Gel Electrophoresis