Please cite this article in press as: O. Fritsch, et al., DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier, DNA Repair (2010), doi:10.1016/j.dnarep.2010.05.003 ARTICLE IN PRESS G Model DNAREP-1386; No. of Pages 10 DNA Repair xxx (2010) xxx–xxx Contents lists available at ScienceDirect DNA Repair journal homepage: www.elsevier.com/locate/dnarepair DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier Olivier Fritsch a,∗,2 , Martin D. Burkhalter b,1,2 , Sanja Kais a , José M. Sogo b , Primo Schär a,∗∗ a Department of Biomedicine, Institute of Biochemistry and Genetics, University of Basel, 4058 Basel, Switzerland b Institute of Cell Biology, Department of Biology, ETH Hönggerberg, 8093 Zürich, Switzerland article info Article history: Received 18 January 2010 Received in revised form 11 May 2010 Accepted 19 May 2010 Available online xxx Keywords: DNA ligase IV Double-strand break ERC Replication fork barrier rDNA abstract DNA double-strand breaks (DSB) were shown to occur at the replication fork barrier in the ribosomal DNA of Saccharomyces cerevisiae using 2D-gel electrophoresis. Their origin, nature and magnitude, however, have remained elusive. We quantified these DSBs and show that a surprising 14% of replicating ribosomal DNA molecules are broken at the replication fork barrier in replicating wild-type cells. This translates into an estimated steady-state level of 7–10 DSBs per cell during S-phase. Importantly, breaks detectable in wild-type and sgs1 mutant cells differ from each other in terms of origin and repair. Breaks in wild- type, which were previously reported as DSBs, are likely an artefactual consequence of nicks nearby the rRFB. Sgs1 deficient cells, in which replication fork stability is compromised, reveal a class of DSBs that are detectable only in the presence of functional Dnl4. Under these conditions, Dnl4 also limits the formation of extrachromosomal ribosomal DNA circles. Consistently, dnl4 cells displayed altered fork structures at the replication fork barrier, leading us to propose an as yet unrecognized role for Dnl4 in the maintenance of ribosomal DNA stability. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Progression of replication forks (RFs) integrates proofing of faithful DNA synthesis and repair or bypass of encountered lesions. RFs are however fragile structures with a potential to collapse at sites of template damage, complex secondary structures, or protein–DNA complexes. Normally, RFs will stall upon encounter- ing such sites and eventually resume replication after the block is removed. If the block persists, however, the RF may collapse and generate a DNA double-strand break (DSB). RF stability is usually studied upon induction of DNA damage [1,2], or under conditions where fork progression is blocked by nucleotide depletion [3,4], both inflicting considerable genomic stress. By contrast, the ribo- somal replication fork barrier (rRFB) in the ribosomal locus (rDNA) Abbreviations: ARS, origin of replication; DSB, double-strand break; EM, electron microscopy; ERC, extrachromosomal rDNA circle; HR, homologous recombination; IGS, intergenic spacer; NHEJ, non-homologous end-joining; rDNA, ribosomal DNA; RF, replication fork; RFB, replication fork barrier; rRFB, ribosomal replication fork barrier; ssDNA, single strand DNA. ∗ Corresponding author. Tel.: +41 61 6953060; fax: +41 61 2673566. ∗∗ Corresponding author. Tel.: +41 61 2670767; fax: +41 61 2673566. E-mail addresses: olivier.fritsch@unibas.ch (O. Fritsch), primo.schaer@unibas.ch (P. Schär). 1 Present address: Institute of Molecular Medicine and Max-Planck Research Group on Stem Cell Aging, University of Ulm, 89081 Ulm, Germany. 2 These authors contributed equally to the work. of Saccharomyces cerevisiae provides a well-characterized “natural” RF pausing model, conserved from yeast to human [5]. The budding yeast rDNA consists of a clustered array of 150–200 repeat units, each carrying the 35S and 5S rRNA genes separated by intergenic spacer regions (IGS1 and IGS2, Fig. 1A). The origin of replication (ARS) in IGS1 is firing when the upstream 35S gene is actively tran- scribed [6,7]. The rRFB element in IGS2 contains one major and two minor barriers [6,8,9] that represent strong pausing sites for RFs in the presence of the Fob1 binding factor [10]. Thus, ARS- initiated replication will freely progress through the 35S gene in the direction of transcription, whereas the opposite RF will stall at the rRFB (Fig. 1A). The latter fork will remain stalled until a RF approaches from an upstream origin, implying that replication ter- mination occurs near rRFB sites, a situation reminiscent of the Ter bacterial system Besides RF pausing, the Fob1-rRFB proteinaceous DNA structure is also implicated in contraction and expansion of the ribosomal array [11,12]. This may involve homologous recombination (HR) as Fob1 was shown to promote HR and DNA DSBs were detected at the rRFB and related to RF pausing and their potential collapse [13–16]. Fork breakage could trigger RAD52-dependent repair in an attempt to re-establish an intact RF [17]. Occasionally, such recom- binational activity will lead to the “pop-out” of rDNA repeat units and, thus, produce extrachromosomal ribosomal circles (ERCs) that accumulate as yeast cells age [18]. Consistently, genetic defects that affect RF progression, such as those impairing the DNA helicases Sgs1 and Rrm3, elevate the rate of recombination genome wide 1568-7864/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2010.05.003