Establishment of a replication fork barrier following induction of DNA binding in mammalian cells

Cell Cycle. 2014;13(10):1607-16. doi: 10.4161/cc.28627. Epub 2014 Mar 25.

Abstract

Understanding the mechanisms that lead to replication fork blocks (RFB) and the means to bypass them is important given the threat that they represent for genome stability if inappropriately handled. Here, to study this issue in mammals, we use integrated arrays of the LacO and/or TetO as a tractable system to follow in time a process in an individual cell and at a single locus. Importantly, we show that induction of the binding by LacI and TetR proteins, and not the presence of the repeats, is key to form the RFB. We find that the binding of the proteins to the arrays during replication causes a prolonged persistence of replication foci at the site. This, in turn, induces a local DNA damage repair (DDR) response, with the recruitment of proteins involved in double-strand break (DSB) repair such as TOPBP1 and 53BP1, and the phosphorylation of H2AX. Furthermore, the appearance of micronuclei and DNA bridges after mitosis is consistent with an incomplete replication. We discuss how the many DNA binding proteins encountered during replication can be dealt with and the consequences of incomplete replication. Future studies exploiting this type of system should help analyze how an RFB, along with bypass mechanisms, are controlled in order to maintain genome integrity.

Keywords: DNA damage repair; DNA replication; LacO-LacI; TetO-TetR; genome instability; replication fork block.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Line, Tumor
  • DNA Breaks, Double-Stranded
  • DNA Repair
  • DNA Replication*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Histones / genetics
  • Histones / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Mice
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • S Phase
  • Tumor Suppressor p53-Binding Protein 1

Substances

  • Carrier Proteins
  • DNA-Binding Proteins
  • H2AX protein, human
  • Histones
  • Intracellular Signaling Peptides and Proteins
  • Nuclear Proteins
  • TOPBP1 protein, human
  • TP53BP1 protein, human
  • Tumor Suppressor p53-Binding Protein 1