Analysis of Replication Dynamics Using the Single-Molecule DNA Fiber Spreading Assay

BMP signaling promotes heart regeneration via alleviation of replication stress

“…Intriguingly, treatment with BMP2 or BMP4 recombinant proteins was sufficient to increase the CldU and IdU track lengths of ongoing replication forks in HSPCs (Figure 8A) , while in U2OS cells only BMP4 had this effect ( Figure 8B ). Replication fork stalling can be identified as tracks where the CldU+ and IdU+ stretches are of different lengths and can be quantified as the ratio between the longer and shorter stretches 37 . Except for a very small change caused by BMP2 in HSPCs (long/short track ratio increased by 3.5% from an average ratio of 1.53 in controls to 1.59 in BMP2 treated cells), BMPs did not affect the frequency at which fork stalling occurred in either HSPCs or U2OS cells ( Supplementary Figure 7A , B ).…”

“…To test whether BMP signaling acts directly on DNA replication, we turned to DNA fiber spreading assays in cultured human cord-blood derived hematopoietic stem and progenitor cells (HSPCs) and the human U2OS cancer cell line. In highly proliferative cells in culture, short incubation with nucleotide analogs (CldU followed by IdU), followed by imaging of individual DNA molecules and quantification of the length of labeled stretches of DNA ("tracks") can reveal several aspects of replication dynamics, including how fast replication forks progress 37 . Both cell types encounter replication challenges even under unperturbed culture conditions, HSPCs due to enforced S-phase entry and U2OS cancer cells due to oncogene overexpression [38][39][40] .…”

S-Phase induced RNAs control ORC1 engagement to H2A.Z and firing of early DNA replication origins

“…In order to examine whether disruption of transcription in the S-Phase, namely reduction of ANCORs ’ levels, would destabilize the formation of the origin and perturb the replication process, we monitored the replication fork dynamics genome-wide by leveraging DNA fiber analysis at single-molecule resolution [41, 42]( Figure 5a ). Synchronized K562 were treated with DRB for three hours upon release into the S-Phase, while sequential additions of 5-iodo-2′-deoxyuridine (IdU) and 5-chloro-2′-deoxyuridine (CldU) [42] were included into the medium to follow the progression of the replication forks during transcriptional inhibition ( Figure 5a ). Upon ANCOR downregulation nasDNA production was impaired and specifically, both DNA replication initiation and fork progressions were found to be decreased in K562 DRB-treated as compared to the control ( Figures 5b,c ).…”