Fig. 4: High-throughput single-molecule approaches for profiling replication dynamics.
From: Monitoring and quantifying replication fork dynamics with high-throughput methods
a HOMARD and ORM are methods of optical mapping of DNA fibers. DNA replication initiations are detected as incorporation stretches of fluorescently labeled dUTP (red) in DNA replication molecules extracted from cells synchronized in G1/S transition and released into S-phase after electroporation with labeling dUTPs (ORM). HOMARD is optimized for analyzing replicated DNA molecules in vitro assays with Xenopus egg extracts. DNA molecules are barcoded and identified through site-specific endonuclease nicking and incorporation of fluorescently labeled dNTPs of a different color (blue). DNA molecules are stained with YoYo-1 (green) imaged with Bionano and aligned using barcoded labels. b DNascent, FORK-seq, and NanoForkSpeed (NFS) methods employ replicative labeling detection by nanopore sequencing. Cells are briefly labeled with BrdU, followed by a short thymidine chase. The ongoing rightward and leftward forks are revealed as a decay of BrdU density during thymidine chase (FORK-seq). The length of the BrdU track divided by labeling time provides individual replication fork speed measurement (NanoForkSpeed). Origins and termini are identified as midpoints of diverging and converging replication tracks, respectively. c Replicon-seq employs targeted cleavage of intact replicons and analysis by Oxford Nanopore sequencing. For this synchronized yeast cells expressing a fusion of replicative helicase subunit MCM4 with MNase are released in S-phase in the presence of BrdU, followed by targeted cleavage of active replicons by adding calcium, DNA purification, and nanopore sequencing. Replicons are detected as symmetric BrdU stretches and fork symmetry is measured as distance to the ARS within the read. d RASAM method. BrdU (purple) is incorporated into nascent DNA, and intact nuclei are incubated with EcoGII m6dA methyltransferase to footprint chromatin accessibility and DNA-protein interactions. Genomic DNA is purified and sequenced using PacBio to detect BrdU and m6dA methylation.
