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

Author(s)

Martins, Davi J. ; Tirman, Stephanie ; Quinet, Annabel ; Menck, Carlos F. M.

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 180, p. 14-pg., 2022-02-01.

Abstract

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the Si nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gapfilling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells. (AU)

• FAPESP's process: 17/05680-0 - Mechanisms of translesion synthesis in human cells
• Grantee: Davi Jardim Martins
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 13/08028-1 - CEGH-CEL - Human Genome and Stem Cell Research Center
• Grantee: Mayana Zatz
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 19/19435-3 - The role of DNA damage and mitochondrial function in vascular, immune and neurological ageing (DNA MoVINg)
• Grantee: Carlos Frederico Martins Menck
• Support Opportunities: Research Projects - Thematic Grants