DNA replication analysis by molecular combing

Analysis of DNA replication by molecular combing was performed, as previously described³⁶. First, asynchronous cells were sequentially labeled with 20 μM IdU for 20 min and 50 μM CldU for 20 min, then chased with 200 μM thymidine for 60–90 min. To preserve long genomic DNA fibers, harvested cells were embedded in low melting point agarose plugs. The plugs were incubated in cell lysis buffer with proteinase K at 50 °C for 16 h, washed 3 times with TE buffer, and then melted in 0.1 M MES (pH 6.5) at 70 °C for 20 min. Agarose was subsequently degraded by adding 2 μl of β-agarase (Biolabs). To stretch DNA fibers, DNA solutions were poured into a Teflon reservoir and DNA was combed onto salinized coverslips (Genomic Vision, cov-002-RUO) using an in-house combing machine. Coverslips were visually examined for DNA density and fiber length by YOYO1 DNA staining (Invitrogen). Combed DNA on coverslips was then baked at 60 °C for 2 h and denatured in 0.5 N NaOH for 20 min. Coverslips were blocked for 10 min in 5% BSA/PBS. IdU, CldU, and single-strand DNA were detected using a mouse antibody directed against BrdU (IgG1, Becton Dickinson, 347580, 1:25 dilution), a rat antibody directed against BrdU (Accurate chemical, OBT0030, 1:200 dilution) and a mouse antibody directed against single-stranded DNA (ssDNA) (IgG 2a, Millipore, MAB3034, 1:100), respectively. Incubation with primary and secondary antibodies were performed at room temperature in 1% BSA in PBS for 1 h and 45 min respectively. The secondary antibodies used were goat anti-mouse Cy3 (Abcam ab6946, 1:100 dilution), goat anti-rat Cy5 (Abcam, ab6565, 1:100 dilution) and goat anti-mouse BV480 (Jackson ImmunoResearch, 115-685-166, 1:50 dilution) for ssDNA. Slides were scanned with a FiberVision Automated Scanner (Genomic Vision). Replication signals on single DNA fibers were analyzed using FiberStudio (Genomic Vision). Only replication signals from high-quality ssDNA (not those from DNA bundles nor those located at the end of a strand) were selected for analyses. Experiments were performed at least in duplicate using independent biological isolations of DNA fibers for each experimental condition. The statistical analyses were performed using Prism 9 (GraphPad software) and the non-parametric Mann–Whitney rank sum test.