Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents

To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of gamma-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of gamma-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced gamma-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.     

Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks

Homologous recombination (HR) deficient cells are sensitive to methyl methanesulfonate (MMS). HR is usually involved in the repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae implying that MMS somehow induces DSBs in vivo. Indeed there is evidence, based on pulsed-field gel electrophoresis (PFGE), that MMS causes DNA fragmentation. However, the mechanism through which MMS induces DSBs has not been demonstrated. Here, we show that DNA fragmentation following MMS treatment, and detected by PFGE is not the consequence of production of cellular DSBs. Instead, DSBs seen following MMS treatment are produced during sample preparation where heat-labile methylated DNA is converted into DSBs. Furthermore, we show that the repair of MMS-induced heat-labile damage requires the base excision repair protein XRCC1, and is independent of HR in both S.cerevisiae and mammalian cells. We speculate that the reason for recombination-deficient cells being sensitive to MMS is due to the role of HR in repair of MMS-induced stalled replication forks, rather than for repair of cellular DSBs or heat-labile damage.     

DNA damage-induced replication arrest in Xenopus egg extracts

Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase alpha, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase alpha and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA.     

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos

S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks.     

Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast

Homologous recombination is believed to play important roles in processing stalled/blocked replication forks in eukaryotes. In accordance with this, recombination is induced by replication fork barriers (RFBs) within the rDNA locus. However, the rDNA locus is a specialised region of the genome, and therefore the action of recombinases at its RFBs may be atypical. We show here for the first time that direct repeat recombination, dependent on Rad22 and Rhp51, is induced by replication fork blockage at a site-specific RFB (RTS1) within a 'typical' genomic locus in fission yeast. Importantly, when the RFB is positioned between the direct repeat, conservative gene conversion events predominate over deletion events. This is consistent with recombination occurring without breakage of the blocked fork. In the absence of the RecQ family DNA helicase Rqh1, deletion events increase dramatically, which correlates with the detection of one-sided DNA double-strand breaks at or near RTS1. These data indicate that Rqh1 acts to prevent blocked replication forks from collapsing and thereby inducing deletion events.     

Homologous recombination prevents methylation-induced toxicity in Escherichia coli

Methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presence of lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single-strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action.     

Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage

The Saccharomyces cerevisiae protein kinase Rad53 plays a key role in maintaining genomic integrity after DNA damage and is an essential component of the ‘intra-S-phase checkpoint’. In budding yeast, alkylating chemicals, such as methyl methanesulfonate (MMS), or depletion of nucleotides by hydroxyurea (HU) stall DNA replication forks and thus activate Rad53 during S-phase. This stabilizes stalled DNA replication forks and prevents the activation of later origins of DNA replication. Here, we report that a reduction in the level of Rad53 kinase causes cells to behave very differently in response to DNA alkylation or to nucleotide depletion. While cells lacking Rad53 are unable to activate the checkpoint response to HU or MMS, so that they rapidly lose viability, a reduction in Rad53 enhances cell survival only after DNA alkylation. This reduction in the level of Rad53 allows S-phase cells to maintain the stability of DNA replication forks upon MMS treatment, but does not prevent the collapse of forks in HU. Our results may have important implications for cancer therapies, as they suggest that partial impairment of the S-phase checkpoint Rad53/Chk2 kinase provides cells with a growth advantage in the presence of drugs that damage DNA.     

Organ-specific effects of DNA methylation by alkylating agents in the inbred Swiss mouse.

Young adult inbred Swiss mice given single or repeated equitoxic doses of N-methyl-N-nitrosourea (MNUA) or methyl methanesulphonate (MMS) develop thymomas and pulmonary adenomas only following MNUA in spite of nearly identical overall alkylation of DNA of tumour target tissues by both agents due mainly to the biologically ineffective product 7-methylguanine. The main difference in DNA alkylation was the production of O6-methylguinine, a known pre-mutagenic product, by MNUA in amounts 10 or more times as large as following MMS. This supports the possibility that somatic mutations are a part of the process of carcinogenesis.     

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

TrAEL-seq provides genome-wide base pair resolution maps of exposed DNA 3’ ends; this reveals replication fork stalling and normal replication profiles in asynchronous, unlabelled wildtype cell populations, along with the sites of resected DNA breaks.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts

Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.     

Endocrine disrupting chemicals modulate expression of O⁶-methylguanine DNA methyltransferase (O⁶-MGMT) gene in the hermaphroditic fish, Kryptolebias marmoratus

O⁶-methylguanine-DNA methyltransferase (O⁶-MGMT; EC 2.1.1.63) is a key repair enzyme that helps to protect the cell against alkylation on DNA by removing a methyl group from the O⁶-position of guanine. Here, we cloned and sequenced the full-length O⁶-MGMT cDNA from the hermaphroditic fish, Kryptolebias marmoratus. Complete Km-O⁶-MGMT cDNA was 1324 bp in length, and the open reading frame of 567 bp encoded a polypeptide of 188 amino acid residues. Phylogenetic analysis revealed that Km-O⁶-MGMT was clustered with those of other fish species. Embryo, juveniles, and aged secondary fish had low levels of Km-O⁶-MGMT mRNA than adults, indicating more susceptibility to DNA damage by alkylating agent exposure during these developmental stages. Km-O⁶-MGMT mRNA levels differed according to tissue type and was highest in the liver. Exposure to an alkylating agent, N-methyl-N-nitrosourea (MNU) exposure increased the mRNA expression of tumor suppressor gene such as p53 and oncogenes such as R-ras1, R-ras3, N-ras, c-fos as well as Km-O⁶-MGMT mRNA in a time-dependent manner. On the contrary, several (anti)estrogenic compounds (17β-estradiol 100 ng/L, tamoxifen 10 μg/L, bisphenol A 600 μg/L, and 4-tert-octylphenol 300 μg/L) suppressed mRNA expression of Km-O⁶-MGMT in most tissues, especially the liver. In juvenile fish, 17β-estradiol, bisphenol A, and 4-tert-octylphenol also decreased the expression of Km-O⁶-MGMT mRNA in a time-dependent manner. Overall, our finding shows that Km-O⁶-MGMT mRNA levels can be modulated by environmental estrogenic compounds as well as alkylating agents. This finding will be helpful to improve our knowledge of the effects of estrogenic compounds that contain the genotoxic ability to inhibit the DNA repair process in aquatic animals.     

Identification of SMARCAL1 as a Component of the DNA Damage Response

SMARCAL1 (also known as HARP) is a SWI/SNF family protein with an ATPase activity stimulated by DNA containing both single-stranded and double-stranded regions. Mutations in SMARCAL1 are associated with the disease Schimke immuno-osseous dysplasia, a multisystem autosomal recessive disorder characterized by T cell immunodeficiency, growth inhibition, and renal dysfunction. The cellular function of SMARCAL1, however, is unknown. Here, using Xenopus egg extracts and mass spectrometry, we identify SMARCAL1 as a protein recruited to double-stranded DNA breaks. SMARCAL1 binds to double-stranded breaks and stalled replication forks in both egg extract and human cells, specifically colocalizing with the single-stranded DNA binding factor RPA. In addition, SMARCAL1 interacts physically with RPA independently of DNA. SMARCAL1 is phosphorylated in a caffeine-sensitive manner in response to double-stranded breaks and stalled replication forks. It has been suggested that stalled forks can be stabilized by a mechanism involving caffeine-sensitive kinases, or they collapse and subsequently recruit Rad51 to promote homologous recombination repair. We show that depletion of SMARCAL1 from U2OS cells leads to increased frequency of RAD51 foci upon generation of stalled replication forks, indicating that fork breakdown is more prevalent in the absence of SMARCAL1. We propose that SMARCAL1 is a novel DNA damage-binding protein involved in replication fork stabilization.     

The novel gene mus7(+) is involved in the repair of replication-associated DNA damage in fission yeast

The progression of replication forks is often impeded by obstacles that cause them to stall or collapse, and appropriate responses to replication-associated DNA damage are important for genome integrity. Here we identified a new gene, mus7(+), that is involved in the repair of replication-associated DNA damage in the fission yeast Schizosaccharomyces pombe. The Deltamus7 mutant shows enhanced sensitivity to methyl methanesulfonate (MMS), camptothecin, and hydroxyurea, agents that cause replication fork stalling or collapse, but not to ultraviolet light or X-rays. Epistasis analysis of MMS sensitivity indicates that Mus7 functions in the same pathway as Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease, which has been implicated in the repair of the replication-associated DNA damage. In Deltamus7 and Deltamus81 cells, the repair of MMS-induced DNA double-strand breaks (DSBs) is severely impaired. Moreover, some cells with either mutation are hyper-elongated or enlarged, and most of these cells accumulate in late G2 phase. Spontaneous Rad22 (recombination mediator protein RAD52 homolog) foci increase in S phase to late G2 phase in Deltamus7 and Deltamus81 cells. These results suggest that replication-associated DSBs accumulate in these cells and that Rad22 foci form in the absence of Mus7 or Mus81. We also found that the rate of spontaneous conversion-type recombination is reduced in mitotic Deltamus7 cells, suggesting that Rhp51- (RAD51 homolog) dependent homologous recombination is disturbed in this mutant. From these data, we propose that Mus7 functions in the repair of replication-associated DSBs by promoting RAD51-dependent conversion-type recombination downstream of Rad22 and Mus81.     

Differential sensitivity of DNA replication and repair in permeable Escherichia coli exposed to various monofunctional methylating or ethylating agents.

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested).The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments.Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity.These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.     

Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability

The minichromosome maintenance (MCM) complex plays essential, conserved roles throughout DNA synthesis: first, as a component of the prereplication complex at origins and, then, as a helicase associated with replication forks. Here we use fission yeast (Schizosaccharomyces pombe) as a model to demonstrate a role for the MCM complex in protecting replication fork structure and promoting recovery from replication arrest. Loss of MCM function generates lethal double-strand breaks at sites of DNA synthesis during replication elongation, suggesting replication fork collapse. MCM function also maintains the stability of forks stalled by hydroxyurea that activate the replication checkpoint. In cells where the checkpoint is activated, Mcm4 binds the Cds1 kinase and undergoes Cds1-dependent phosphorylation. MCM proteins also interact with proteins involved in homologous recombination, which promotes recovery from arrest by ensuring normal mitosis. We suggest that the MCM complex links replication fork stabilization with checkpoint arrest and recovery through direct interactions with checkpoint and recombination proteins and that this role in S-phase genome stability is conserved from yeast to human cells.     

Low levels of DNA polymerase alpha induce mitotic and meiotic instability in the ribosomal DNA gene cluster of Saccharomyces cerevisiae

The ribosomal DNA (rDNA) genes of Saccharomyces cerevisiae are located in a tandem array of about 150 repeats. Using a diploid with markers flanking and within the rDNA array, we showed that low levels of DNA polymerase alpha elevate recombination between both homologues and sister chromatids, about five-fold in mitotic cells and 30-fold in meiotic cells. This stimulation is independent of Fob1p, a protein required for the programmed replication fork block (RFB) in the rDNA. We observed that the fob1 mutation alone significantly increased meiotic, but not mitotic, rDNA recombination, suggesting a meiosis-specific role for this protein. We found that meiotic cells with low polymerase alpha had decreased Sir2p binding and increased Spo11p-catalyzed double-strand DNA breaks in the rDNA. Furthermore, meiotic crossover interference in the rDNA is absent. These results suggest that the hyper-Rec phenotypes resulting from low levels of DNA polymerase alpha in mitosis and meiosis reflect two fundamentally different mechanisms: the increased mitotic recombination is likely due to increased double-strand DNA breaks (DSBs) resulting from Fob1p-independent stalled replication forks, whereas the hyper-Rec meiotic phenotype results from increased levels of Spo11-catalyzed DSBs in the rDNA.     

Proficient repair in chromatin remodeling defective ino80 mutants of Saccharomyces cerevisiae highlights replication defects as the main contributor to DNA damage sensitivity

Ino80 is an evolutionarily conserved member of the SWI2/SNF2-family of ATPases in Saccharomyces cerevisiae. It resides in a multiprotein helicase/chromatin remodeling complex, and has been shown to play a key role in the stability of replication forks during replication stress. Though yeast with defects in ino80 show sensitivity to killing by a variety of DNA-damaging agents, a role for the INO80 protein complex in the repair of DNA has only been assessed for double-strand breaks, and the results are contradictory and inconclusive. We report that ino80Δ cells are hypersensitive to DNA base lesions induced by ultraviolet (UV) radiation and methyl methanesulfonate (MMS), but show little (or no) increased sensitivity to the DNA double-strand break (DSB)-inducing agents ionizing radiation and camptothecin. Importantly, ino80Δ cells display efficient removal of UV-induced cyclobutane pyrimidine dimers, and show a normal rate of removal of DNA methylation damage after MMS exposure. In addition, ino80Δ cells have an overall normal rate of repair of DSBs induced by ionizing radiation. Altogether, our data support a model of INO80 as an important suppressor of genome instability in yeast involved in DNA damage tolerance through a role in stability and recovery of broken replication forks, but not in the repair of lesions leading to such events. This conclusion is in contrast to strong evidence for the DNA repair-promoting role of the corresponding INO80 complexes in higher eukaryotes. Thus, our results provide insight into the specialized roles of the INO80 subunits and the differential needs of different species for chromatin remodeling complexes in genome maintenance.