Electron Microscopy of Replication Intermediates

Electron microscopy analysis of the replication intermediates was performed as previously described^{7 (link)}. Briefly, replication reactions were stopped at 90 min with 10 volumes of Stop Solution C (100 mM Tris-HCl [pH 7.5], 6.7 mM MgCl₂, 1 mM EDTA [pH 8.0], and 1% SDS). The DNA was crosslinked with trimethylpsoralen (Sigma) and irradiation with UV light at 365 nM prior to protein extraction and DNA purification. Purified DNA was incubated with E. coli single-stranded DNA binding protein (SSB), fixed with 0.3% glutaraldehyde, then purified by size-exclusion chromatography. Eluted complexes were mounted onto grids, which were then subjected to rotary shadowing with platinum and carbon coating using a Leica Ace600 coating system. Samples were imaged using a JEOL 1200EX transmission electron microscope equipped with a 2k CCD camera (Advanced Microscopy Techniques). After blinding the scorer to the conditions, reversed forks were counted and expressed as a percentage of pre-incision structures, which was then normalized to the mock-depleted condition.