title: Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair.
abstract: Replication origins are under tight regulation to ensure activation occurs only once per cell cycle [1, 2]. 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 [2-7]. If the checkpoint is blocked, cells enter mitosis with partially re-replicated DNA that generates chromosome breaks and fusions [5]. 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 [3]. 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 [8-10], 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) [10-12]. Precise developmental timing of DAFC origin firing permits identification of forks at defined points after origin initiation [13, 14]. 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.
authors.affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 68-132, Cambridge, MA 02139, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Alexander
authors.affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
authors.forename: M Inmaculada
authors.initials: MI
authors.lastname: Barrasa
authors.affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 68-132, Cambridge, MA 02139, USA. Electronic address: weaver@wi.mit.edu.
authors.forename: Terry L
authors.initials: TL
authors.lastname: Orr-Weaver
mesh_terms: D000818:Animals; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004261:DNA Replication; D004331:Drosophila melanogaster / Q000235:genetics*