The rarA gene as part of an expanded RecFOR recombination pathway: Negative epistasis and synthetic lethality with ruvB,recG, and recQ

The RarA protein, homologous to human WRNIP1 and yeast MgsA, is a AAA⁺ ATPase and one of the most highly conserved DNA repair proteins. With an apparent role in the repair of stalled or collapsed replication forks, the molecular function of this protein family remains obscure. Here, we demonstrate that RarA acts in late stages of recombinational DNA repair of post-replication gaps. A deletion of most of the rarA gene, when paired with a deletion of ruvB or ruvC, produces a growth defect, a strong synergistic increase in sensitivity to DNA damaging agents, cell elongation, and an increase in SOS induction. Except for SOS induction, these effects are all suppressed by inactivating recF, recO, or recJ, indicating that RarA, along with RuvB, acts downstream of RecA. SOS induction increases dramatically in a rarA ruvB recF/O triple mutant, suggesting the generation of large amounts of unrepaired ssDNA. The rarA ruvB defects are not suppressed (and in fact slightly increased) by recB inactivation, suggesting RarA acts primarily downstream of RecA in post-replication gaps rather than in double strand break repair. Inactivating rarA, ruvB and recG together is synthetically lethal, an outcome again suppressed by inactivation of recF, recO, or recJ. A rarA ruvB recQ triple deletion mutant is also inviable. Together, the results suggest the existence of multiple pathways, perhaps overlapping, for the resolution or reversal of recombination intermediates created by RecA protein in post-replication gaps within the broader RecF pathway. One of these paths involves RarA.     

Activation of the DNA damage checkpoint in mutants defective in DNA replication initiation

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.     

Functional overlap between the structure-specific nucleases Yen1 and Mus81-Mms4 for DNA-damage repair in S. cerevisiae

In eukaryotic cells, multiple DNA repair mechanisms respond to a wide variety of DNA lesions. Homologous recombination-dependent repair provides a pathway for dealing with DNA double-strand breaks and replication fork demise. A key step in this process is the resolution of recombination intermediates such as Holliday junctions (HJs). Recently, nucleases from yeast (Yen1) and human cells (GEN1) were identified that can resolve HJ intermediates, in a manner analogous to the E. coli HJ resolvase RuvC. Here, we have analyzed the role of Yen1 in DNA repair in S. cerevisiae, and show that while yen1Δ mutants are repair-proficient, yen1Δ mus81Δ double mutants are exquisitely sensitive to a variety of DNA-damaging agents that disturb replication fork progression. This phenotype is dependent upon RAD52, indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81. After MMS treatment, yen1Δ mus81Δ double mutants arrest with a G2 DNA content and unsegregated chromosomes. These findings indicate that Yen1 can act upon recombination/repair intermediates that arise in MUS81-defective cells following replication fork damage.     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Deletion of ULS1 confers damage tolerance in sgs1 mutants through a Top3-dependent D-loop mediated fork restart pathway

Homologous recombination (HR)-based repair during DNA replication can apparently utilize several partially overlapping repair pathways in response to any given lesion. A key player in HR repair is the Sgs1-Top3-Rmi1 (STR) complex, which is critical for resolving X-shaped recombination intermediates formed following bypass of methyl methanesulfonate (MMS)-induced damage. STR mutants are also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU), but unlike MMS treatment, HU treatment is not accompanied by X-structure accumulation, and it is thus unclear how STR functions in this context. Here we provide evidence that HU-induced fork stalling enlists Top3 prior to recombination intermediate formation. The resistance of sgs1Δ mutants to HU is enhanced by the absence of the putative SUMO (Small Ubiquitin MOdifier)-targeted ubiquitin ligase, Uls1, and we demonstrate that Top3 is required for this enhanced resistance and for coordinated breaks and subsequent d-loop formation at forks stalled at the ribosomal DNA (rDNA) replication fork block (RFB). We also find that HU resistance depends on the catalytic activity of the E3 SUMO ligase, Mms21, and includes a rapid Rad51-dependent restart mechanism that is different from the slow Rad51-independent HR fork restart mechanism operative in sgs1Δ ULS1+ mutants. These data support a model in which repair of HU-induced damage in sgs1Δ mutants involves an error-prone break-induced replication pathway but, in the absence of Uls1, shifts to one that is higher-fidelity and involves the formation of Rad51-dependent d-loops.     

Replication arrest is a major threat to growth at low temperature in Antarctic Pseudomonas syringae Lz4W

Chromosomal damage was detected previously in the recBCD mutants of the Antarctic bacterium Pseudomonas syringae Lz4W, which accumulated linear chromosomal DNA leading to cell death and growth inhibition at 4°C. RecBCD protein generally repairs DNA double‐strand breaks by RecA‐dependent homologous recombination pathway. Here we show that ΔrecA mutant of P. syringae is not cold‐sensitive. Significantly, inactivation of additional DNA repair genes ruvAB rescued the cold‐sensitive phenotype of ΔrecBCD mutant. The ΔrecA and ΔruvAB mutants were UV‐sensitive as expected. We propose that, at low temperature DNA replication encounters barriers leading to frequent replication fork (RF) arrest and fork reversal. RuvAB binds to the reversed RFs (RRFs) having Holliday junction‐like structures and resolves them upon association with RuvC nuclease to cause linearization of the chromosome, a threat to cell survival. RecBCD prevents this by degrading the RRFs, and facilitates replication re‐initiation. This model is consistent with our observation that low temperature‐induced DNA lesions do not evoke SOS response in P. syringae. Additional studies show that two other repair genes, radA (encoding a RecA paralogue) and recF are not involved in providing cold resistance to the Antarctic bacterium.     

RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.     

Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression

Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.     

FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine

Fanconi anemia (FA) is a rare hereditary disorder caused by mutations in any one of the FANC genes. FA cells are mainly characterized by extreme hypersensitivity to interstrand crosslink (ICL) agents. Additionally, the FA proteins play a crucial role in concert with homologous recombination (HR) factors to protect stalled replication forks. Here, we report that the 5-methyl-2’-deoxycytidine (5mdC) demethylation (pathway) intermediate 5-hydroxymethyl-2’-deoxycytidine (5hmdC) and its deamination product 5-hydroxymethyl-2’-deoxyuridine (5hmdU) elicit a DNA damage response, chromosome aberrations, replication fork impairment and cell viability loss in the absence of FANCD2. Interestingly, replication fork instability by 5hmdC or 5hmdU was associated to the presence of Poly(ADP-ribose) polymerase 1 (PARP1) on chromatin, being both phenotypes exacerbated by olaparib treatment. Remarkably, Parp1^−/− cells did not show any replication fork defects or sensitivity to 5hmdC or 5hmdU, suggesting that retained PARP1 at base excision repair (BER) intermediates accounts for the observed replication fork defects upon 5hmdC or 5hmdU incorporation in the absence of FANCD2. We therefore conclude that 5hmdC is deaminated in vivo to 5hmdU, whose fixation by PARP1 during BER, hinders replication fork progression and contributes to genomic instability in FA cells.Subject terms: DNA damage and repair, DNA replication     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells

The SOS response is a DNA damage response pathway that serves as a general safeguard of genome integrity in bacteria. Extensive studies of the SOS response in Escherichia coli have contributed to establishing the key concepts of cellular responses to DNA damage. However, how the SOS response impacts on the dynamics of DNA replication fork movement remains unknown. We found that inducing the SOS response decreases the mean speed of individual replication forks by 30–50% in E. coli cells, leading to a 20–30% reduction in overall DNA synthesis. dinB and recA belong to a group of genes that are upregulated during the SOS response, and encode the highly conserved proteins DinB (also known as DNA polymerase IV) and RecA, which, respectively, specializes in translesion DNA synthesis and functions as the central recombination protein. Both genes were independently responsible for the SOS-dependent slowdown of replication fork progression. Furthermore, fork speed was reduced when each gene was ectopically expressed in SOS-uninduced cells to the levels at which they are expressed in SOS-induced cells. These results clearly indicate that the increased expression of dinB and recA performs a novel role in restraining the progression of an unperturbed replication fork during the SOS response.     

The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication

The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1⁺, two temperature-sensitive mcb1 gene mutants (mcb1^ts) were isolated. Extensive genetic analysis showed that the mcb1^ts mutants were suppressed by a mcm5⁺ multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1^ts mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1^ts mutants. Furthermore, the mcb1^ts mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex.     

Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase

Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1^−/− chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1^−/− cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3^−/− DT40 cells. Rather, we observed an increased number of replication fibers in Chk1^−/− cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1^−/− cells are associated with the accumulation of aberrant replication fork structures.     

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork “collapse point” in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.     

Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G₁ phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.     

The archaeal Xpf/Mus81/FANCM homolog Hef and the Holliday junction resolvase Hjc define alternative pathways that are essential for cell viability in Haloferax volcanii

The XPF/MUS81 family of endonucleases is found in eukaryotes and archaea, in the former they play a critical role in DNA repair and replication fork restart. Hef is a XPF/MUS81 family member found in Euryarchaea and is related to the Fanconi anemia protein FANCM. We have studied the role of Hef in the euryarchaeon Haloferax volcanii. Unlike Xpf in eukaryotes, Hef is not involved in nucleotide excision repair; instead, this function is encoded by the uvrABC genes. Similarly, deletion of hef confers only moderate sensitivity to DNA crosslinking agents, whereas mutation of FANCM in leads to hypersensitivity in eukaryotes. However, Hef is essential for cell viability when the Holliday junction resolvase Hjc is absent, and both the helicase and nuclease activities of Hef are indispensable. By contrast, single mutants of hjc and hef display no significant defects in growth or homologous recombination. This suggests that Hef and Hjc are redundant for the resolution of recombination intermediates, and that Hef is the functional homolog of eukaryotic Mus81. Furthermore, deletion of hef in a recombination-deficient ΔradA background is highly deleterious but deletion of hjc has no effect. Therefore, Hjc acts exclusively in homologous recombination whereas Hef, in addition to its role in resolving recombination intermediates, can act in a pathway that avoids the use of homologous recombination. We propose that Hef and Hjc provide alternative means to restart stalled DNA replication forks.     

Role of Lamin B1 in Chromatin Instability

Nuclear lamins play important roles in the organization and structure of the nucleus; however, the specific mechanisms linking lamin structure to nuclear functions are poorly defined. We demonstrate that reducing nuclear lamin B1 expression by short hairpin RNA-mediated silencing in cancer cell lines to approximately 50% of normal levels causes a delay in the cell cycle and accumulation of cells in early S phase. The S phase delay appears to be due to the stalling and collapse of replication forks. The double-strand DNA breaks resulting from replication fork collapse were inefficiently repaired, causing persistent DNA damage signaling and the assembly of extensive repair foci on chromatin. The expression of multiple factors involved in DNA replication and repair by both nonhomologous end joining and homologous repair is misregulated when lamin B1 levels are reduced. We further demonstrate that lamin B1 interacts directly with the promoters of some genes associated with DNA damage response and repair, including BRCA1 and RAD51. Taken together, the results suggest that the maintenance of lamin B1 levels is required for DNA replication and repair through regulation of the expression of key factors involved in these essential nuclear functions.     

Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase.

A cell extract prepared from the lig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin E1 plasmid at 25 °C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from the thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25 °C to 32 °C, whereas that in the lig⁺ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the lig-ts extract, even at 25 °C.The DNA pulse-labelled with the lig-ts extract for 30 seconds at 32 °C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular molecules upon chasing with a lig⁺ extract. DNA-DNA hybridization experiments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5′-to-3′ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component.The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plasmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compound does not interfere with elongation of progeny strands. Chain elongation in the lig-ts extract at 25 °C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the lig-ts extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin E1 plasmid DNA replicate discontinuously.     

Replication Fork Stalling and Checkpoint Activation by a PKD1 Locus Mirror Repeat Polypurine-Polypyrimidine (Pu-Py) Tract

DNA sequences prone to forming noncanonical structures (hairpins, triplexes, G-quadruplexes) cause DNA replication fork stalling, activate DNA damage responses, and represent hotspots of genomic instability associated with human disease. The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat tract from the human polycystic kidney disease (PKD1) intron 21 forms non-B DNA secondary structures in vitro. We show that the PKD1 mirror repeat also causes orientation-dependent fork stalling during replication in vitro and in vivo. When integrated alongside the c-myc replicator at an ectopic chromosomal site in the HeLa genome, the Pu-Py mirror repeat tract elicits a polar replication fork barrier. Increased replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) checkpoint protein binding near the mirror repeat sequence suggests that the DNA damage response is activated upon replication fork stalling. Moreover, the proximal c-myc origin of replication was not required to cause orientation-dependent checkpoint activation. Cells expressing the replication fork barrier display constitutive Chk1 phosphorylation and continued growth, i.e. checkpoint adaptation. Excision of the Pu-Py mirror repeat tract abrogates the DNA damage response. Adaptation to Chk1 phosphorylation in cells expressing the replication fork barrier may allow the accumulation of mutations that would otherwise be remediated by the DNA damage response.