Replication Fork Progression during Re-replication Requires the DNA Damage Checkpoint and Double-Strand Break Repair
Author(s)Barrasa, M. Inmaculada; Orr-Weaver, Terry; Alexander, Jessica Lynne
MetadataShow full item record
Replication origins are under tight regulation to ensure activation occurs only once per cell cycle. Origin re-firing in a single S phase leads to the generation of DNA double-strand breaks (DSBs) and activation of the DNA damage checkpoint. If the checkpoint is blocked, cells enter mitosis with partially re-replicated DNA that generates chromosome breaks and fusions. These types of chromosomal aberrations are common in numerous human cancers, suggesting that re-replication events contribute to cancer progression. It was proposed that fork instability and DSBs formed during re-replication are the result of head-to-tail collisions and collapse of adjacent replication forks. However, previously studied systems lack the resolution to determine whether the observed DSBs are generated at sites of fork collisions. Here, we utilize the Drosophila ovarian follicle cells, which exhibit re-replication under precise developmental control, to model the consequences of re-replication at actively elongating forks. Re-replication occurs from specific replication origins at six genomic loci, termed Drosophila amplicons in follicle cells (DAFCs). Precise developmental timing of DAFC origin firing permits identification of forks at defined points after origin initiation. Here, we show that DAFC re-replication causes fork instability and generates DSBs at sites of potential fork collisions. Immunofluorescence and ChIP-seq demonstrate the DSB marker γH2Av is enriched at elongating forks. Fork progression is reduced in the absence of DNA damage checkpoint components and nonhomologous end-joining (NHEJ), but not homologous recombination. NHEJ appears to continually repair forks during re-replication to maintain elongation.
DepartmentMassachusetts Institute of Technology. Department of Biology; Whitehead Institute for Biomedical Research
Alexander, Jessica L., M. Inmaculada Barrasa, and Terry L. Orr-Weaver. “Replication Fork Progression during Re-Replication Requires the DNA Damage Checkpoint and Double-Strand Break Repair.” Current Biology 25.12 (2015): 1654–1660.
Author's final manuscript