Single cell analysis of human RAD18-dependent DNA post-replication repair by alkaline bromodeoxyuridine comet assay

PLoS One. 2013 Aug 6;8(8):e70391. doi: 10.1371/journal.pone.0070391. Print 2013.

Abstract

Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bromodeoxyuridine / metabolism
  • Comet Assay / methods*
  • DNA / biosynthesis*
  • DNA / genetics*
  • DNA Damage / genetics
  • DNA Repair* / radiation effects
  • DNA Replication* / radiation effects
  • DNA-Binding Proteins / deficiency
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Dose-Response Relationship, Radiation
  • Gene Knockout Techniques
  • HCT116 Cells
  • HeLa Cells
  • Humans
  • Kinetics
  • S Phase / genetics
  • S Phase / radiation effects
  • Single-Cell Analysis / methods*
  • Ubiquitin-Protein Ligases
  • Ultraviolet Rays

Substances

  • DNA-Binding Proteins
  • RAD18 protein, human
  • DNA
  • Ubiquitin-Protein Ligases
  • Bromodeoxyuridine

Grant support

This work was supported by the Hungarian Science Foundation grants (OTKA 101225, GOP-1.1.1-11-2012-0030 and GOP-1.1.1-11-2012-0384), and IPA Cross-border Co-operation Programme (HUSRB/1002/214/126). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.