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Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay
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JoVE Journal Genetics
Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

DOI:
10.3791/63448-v

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10:32 min

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February 03, 2022

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, , ,
1Department of Microbiology, Institute of Biomedical Sciences,University of São Paulo, 2Division of Oncology, Department of Internal Medicine,Washington University in St. Louis

Chapters

  • 00:04Introduction
  • 01:27S1 Fiber Experimental Setup
  • 03:51DNA Fiber Preparation by Spreading
  • 04:58Immunostaining of DNA Fibers
  • 06:45Image Acquisition and Analysis
  • 08:39Results: Post-Replicative ssDNA Gaps on Ongoing Forks by S1 Fiber Assay and Post-Replicative Repair by Gap Filling
  • 09:42Conclusion

Summary

Automatic Translation

Automatic Translation

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

Tags

DNA Fiber AssayPost-replicative GapsS1 NucleaseDNA RepairReplication StressGenome IntegrityUV-C IrradiationCldU IncorporationCSK PermeabilizationDNA Lesion Detection

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