Interference in DNA replication can cause mitotic chromosomal breakage unassociated with double-strand breaks

PLoS One. 2013;8(4):e60043. doi: 10.1371/journal.pone.0060043. Epub 2013 Apr 3.

Abstract

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54(-/-/)KU70(-/-) DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54(-/-/)LIG4(-/-) Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Nuclear / genetics
  • Aphidicolin / pharmacology
  • Apoptosis
  • Cell Line, Tumor
  • Chickens
  • Chromosome Breakage*
  • DNA Breaks, Double-Stranded
  • DNA Helicases / physiology
  • DNA Repair
  • DNA Repair Enzymes / physiology
  • DNA Replication / drug effects*
  • DNA-Binding Proteins / genetics
  • Fluorouracil / pharmacology
  • Gene Knockout Techniques
  • Humans
  • Hydroxyurea / pharmacology
  • Ku Autoantigen
  • Mitosis*

Substances

  • Antigens, Nuclear
  • DNA-Binding Proteins
  • Aphidicolin
  • DNA Helicases
  • Xrcc6 protein, human
  • Ku Autoantigen
  • DNA Repair Enzymes
  • Fluorouracil
  • Hydroxyurea

Grant support

This work was supported by the Environment Research and Technology Development Fund (RF-1005) of the Ministry of the Environment, Japan and a research grant from CERI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.