p53 prevents the accumulation of double-strand DNA breaks at stalled-replication forks induced by UV in human cells

Shoshana Squires, Julia A. Coates, Michal Goldberg, Lorraine H. Toji, Stephen P. Jackson, Duncan J. Clarke, Robert T. Johnson

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

To investigate the mechanism by which UV irradiation causes S-phase-dependent chromosome aberrations and thereby genomic instability, we have developed an assay to study the DNA structure of replication forks (RFs) in UV-irradiated mammalian cells, using pulse-field gel electrophoresis for the DNA analysis. We demonstrate that replication stalling at UV-induced pyrimidine dimers results in the formation of single-strand DNA (ssDNA) regions and incomplete RF structures. In normal and in nucleotide-excision-repair (NER)-defective xeroderma pimentosum (XP) cells, stalling at dimers is rapid and prolonged and recovery depends on dimer repair or bypass. By contrast, XP variant (XPV) cells, defective in replication of a UV-damaged template due to mutation of bypass-polymerase η, fail to arrest at dimers, resulting in a much higher frequency of ssDNA regions in the stalled RFs. We show that the stability of UV-arrested RFs depends directly on functional p53, and indirectly on NER and pol η. In p53-deficient cells, the stalled sites give rise to double-strand DNA breaks (DSBs), at a frequency inversely correlated with the repair capacity of the cell. In normal cells only a fraction of the stalled sites give rise to DSBs, while in XPASV, XPDSV and also XPVSV, all the sites do. XPVSV cells, although repair proficient, accumulate almost double the number of DSBs, suggesting that a high frequency of ssDNA regions in UV-arrested forks cause RF instability. These replication-associated DSBs do not accumulate in p53-proficient human cells. We propose that a major mechanism by which p53 maintains genome stability is the prevention of DSB accumulation at long-lived ssDNA regions in stalled-replication forks.

Original languageEnglish (US)
Pages (from-to)1543-1557
Number of pages15
JournalCell Cycle
Volume3
Issue number12
DOIs
StatePublished - Dec 2004

Keywords

  • Double strand breaks
  • Genome stability
  • Stalled replication forks
  • UV damage
  • XP
  • XPV
  • p53

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