DNA fiber spreading assay is an invaluable technique to visualize and follow the spatial and temporal progress of individual DNA replication forks. It provides information on the DNA replication progress and its regulation under normal conditions as well as on replication stress induced by environmental...
DNA fiber spreading assay to test HDACi effects on DNA and its replication. Methods Mol. Biol. 1510, 103–113 (2017). Article CAS PubMed Google Scholar Johnson, G. E. Mammalian cell HPRT gene mutation assay: test methods. Methods Mol. Biol. 817, 55–67 (2012). Article CAS PubMed ...
DNA fiber spreading assay To measure DNA replication track lengths for assessment of replication speed the DNA fiber spreading technique was performed as detailed in [20]. CldU (5-Chloro-2-deoxyuridine, Sigma-Aldrich by Merck, Darmstadt, Germany) was incorporated for 20 min. After washing, IdU...
DNA Fiber Spreading Assay to Test HDACi Effects on DNA and Its Replication Chapter © 2017 Assessing Cell Cycle Independent Function of the CDK Inhibitor p21CDKN1A in DNA Repair Chapter © 2016 MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation ...
Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. 2020 Wiley Periodicals LLC. Basic Protocol 1: DNA fiber labeling Basic Protocol 2: DNA fiber spreading Basic Protocol 3: ...
In support of this, we performed a DNA fiber assay at single-molecule resolution and showed that knockdown of KILR using two independent crRNAs promotes proliferation by increasing the speed of DNA replication (Fig. 3b). More recently, it was shown that the RPA complex can stabilize the DNA...
DNA Fiber Assay DNA fibers were prepared as described previously (Chen et al., 2015). Cells were pulsed with 50 μM IdU and 50 μM CldU for 20 minutes each for MDA-MB-231 and 30 minutes each for Hs578t and MCF7 cells. Cells were then harvested, washed in PBS, and resuspended at...
DNA fiber analysis Cells were pulse labeled with 25 μM CldU followed by 250 μM IdU for 20–45 min each. Labeled cells were trypsinized, lysed in spreading buffer (200 mM Tris-Hcl pH 7.4, 50 mM EDTA, and 0.5% SDS) and spread on microscope slide (Menzel-Gläser, Supe...
To test the role of PFN1 in DNA replication, we next performed single-molecule DNA fiber assay by pulsing cells with IdU and CIdU sequentially47. By quantifying the tract lengths of dual-colored fibers, we found that PFN1 knockdown significantly decreased DNA replication speed in multiple human...
aStrategy for the S1-modified DNA fiber assay with ultraviolet (UV-C) irradiation.bCldU/IdU ratios after S1-modified DNA fiber assay in cells treated with 5 J/m2UV-C or mock-treated as in (a), in the presence of nonT or PrimPol siRNA. Lines represent mean. Data from 4 repeats.c...