SUMO protease and proteasome recruitment at the nuclear periphery differently affect replication dynamics at arrested forks.

Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for the repair of DNA damage. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function is not alleviated by preventing SUMO chain formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect the dynamics of RDR in distinct ways. Our work probes two distinct mechanisms by which the NPC environment ensures optimal RDR, both controlled by different NPC components.

Schizosaccharomyces pombe Rtf2 is important for replication fork barrier activity of RTS1 via splicing of Rtf1.

Arrested replication forks, when restarted by homologous recombination, result in error-prone DNA syntheses and non-allelic homologous recombination. Fission yeast RTS1 is a model fork barrier used to probe mechanisms of recombination-dependent restart. RTS1 barrier activity is entirely dependent on the DNA binding protein Rtf1 and partially dependent on a second protein, Rtf2. Human RTF2 was recently implicated in fork restart, leading us to examine fission yeast Rtf2's role in more detail. In agreement with previous studies, we observe reduced barrier activity upon rtf2 deletion. However, we identified Rtf2 to be physically associated with mRNA processing and splicing factors and rtf2 deletion to cause increased intron retention. One of the most affected introns resided in the rtf1 transcript. Using an intronless rtf1, we observed no reduction in RFB activity in the absence of Rtf2. Thus, Rtf2 is essential for correct rtf1 splicing to allow optimal RTS1 barrier activity.

Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast.

Efficient replication of terminal DNA is crucial to maintain telomere stability. In fission yeast, Taz1 and the Stn1-Ten1 (ST) complex play prominent roles in DNA-ends replication. However, their function remains elusive. Here, we have analyzed genome-wide replication and show that ST does not affect genome-wide replication but is crucial for the efficient replication of a subtelomeric region called STE3-2. We further show that, when ST function is compromised, a homologous recombination (HR)-based fork restart mechanism becomes necessary for STE3-2 stability. While both Taz1 and Stn1 bind to STE3-2, we find that the STE3-2 replication function of ST is independent of Taz1 but relies on its association with the shelterin proteins Pot1-Tpz1-Poz1. Finally, we demonstrate that the firing of an origin normally inhibited by Rif1 can circumvent the replication defect of subtelomeres when ST function is compromised. Our results help illuminate why fission yeast telomeres are terminal fragile sites.

Replication dynamics of recombination-dependent replication forks.

Replication forks restarted by homologous recombination are error prone and replicate both strands semi-conservatively using Pol δ. Here, we use polymerase usage sequencing to visualize in vivo replication dynamics of HR-restarted forks at an S. pombe replication barrier, RTS1, and model replication by Monte Carlo simulation. We show that HR-restarted forks synthesise both strands with Pol δ for up to 30 kb without maturing to a δ/ε configuration and that Pol α is not used significantly on either strand, suggesting the lagging strand template remains as a gap that is filled in by Pol δ later. We further demonstrate that HR-restarted forks progress uninterrupted through a fork barrier that arrests canonical forks. Finally, by manipulating lagging strand resection during HR-restart by deleting pku70, we show that the leading strand initiates replication at the same position, signifying the stability of the 3' single strand in the context of increased resection.

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes.

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

Analysis of Replicative Polymerase Usage by Ribonucleotide Incorporation.

Mapping the usage of replicative DNA polymerases has previously proved to be technically challenging. By exploiting mutant polymerases that incorporate ribonucleotides into the DNA with a significantly higher proficiency than their wild-type counterparts, we and others have developed methods that can identify what proportion of each DNA strand (i.e., the Watson and Crick strands) is replicated by a specific DNA polymerase. The incorporation of excess ribonucleotides by a mutated polymerase effectively marks, in each individual cells, the DNA strand that is replicated by that specific mutated polymerase. Changes to DNA polymerase usage can be examined at specific loci by Southern blot analysis while a global analysis of polymerase usage can be achieved by applying next-generation sequencing. This genome-wide data also provides a direct measure of replication origin efficiency and can be used to indirectly calculate replication timing.

A defect in homologous recombination leads to increased translesion synthesis in E. coli.

DNA damage tolerance pathways allow cells to duplicate their genomes despite the presence of replication blocking lesions. Cells possess two major tolerance strategies, namely translesion synthesis (TLS) and homology directed gap repair (HDGR). TLS pathways involve specialized DNA polymerases that are able to synthesize past DNA lesions with an intrinsic risk of causing point mutations. In contrast, HDGR pathways are essentially error-free as they rely on the recovery of missing information from the sister chromatid by RecA-mediated homologous recombination. We have investigated the genetic control of pathway choice between TLS and HDGR in vivo in Escherichia coli In a strain with wild type RecA activity, the extent of TLS across replication blocking lesions is generally low while HDGR is used extensively. Interestingly, recA alleles that are partially impaired in D-loop formation confer a decrease in HDGR and a concomitant increase in TLS. Thus, partial defect of RecA's capacity to invade the homologous sister chromatid increases the lifetime of the ssDNA.RecA filament, i.e. the 'SOS signal'. This increase favors TLS by increasing both the TLS polymerase concentration and the lifetime of the TLS substrate, before it becomes sequestered by homologous recombination. In conclusion, the pathway choice between error-prone TLS and error-free HDGR is controlled by the efficiency of homologous recombination.

Chronology in lesion tolerance gives priority to genetic variability.

The encounter of a replication fork with a blocking DNA lesion is a common event that cells need to address properly to preserve genome integrity. Cells possess two main strategies to tolerate unrepaired lesions: potentially mutagenic translesion synthesis (TLS) and nonmutagenic damage avoidance (DA). Little is known about the partitioning between these two strategies. Because genes involved in DA mechanisms (i.e., recA) are expressed early and genes involved in TLS (i.e., Pol V) are expressed late during the bacterial SOS response, it has long been thought that TLS was the last recourse to bypass DNA lesions when repair and nonmutagenic DA mechanisms have failed. By using a recently described methodology, we followed the fate of a single replication-blocking lesion introduced in the Escherichia coli genome during acute genotoxic stress. We show that lesion tolerance events (i) only occur when the SOS response is fully induced and (ii) are executed in chronological order, with TLS coming first, followed by DA. Therefore, in response to genotoxic stress, bacterial cells give priority to TLS, a minor pathway able to generate genetic diversity before implementing the major nonmutagenic pathway that ensures survival.

Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome.

Although most deoxyribonucleic acid (DNA) lesions are accurately repaired before replication, replication across unrepaired lesions is the main source of point mutations. The lesion tolerance processes, which allow damaged DNA to be replicated, entail two branches, error-prone translesion synthesis (TLS) and error-free damage avoidance (DA). While TLS pathways are reasonably well established, DA pathways are poorly understood. The fate of a replication-blocking lesion is generally explored by means of plasmid-based assays. Although such assays represent efficient tools to analyse TLS, we show here that plasmid-borne lesions are inappropriate models to study DA pathways due to extensive replication fork uncoupling. This observation prompted us to develop a method to graft, site-specifically, a single lesion in the genome of a living cell. With this novel assay, we show that in Escherichia coli DA events massively outweigh TLS events and that in contrast to plasmid, chromosome-borne lesions partially require RecA for tolerance.

Formation, persistence, and identification of DNA adducts formed by the carcinogenic environmental pollutant o-anisidine in rats.

2-Methoxyaniline (o-anisidine) is an industrial and environmental pollutant causing tumors of urinary bladder in rodents. Here, we investigated the formation and persistence of DNA adducts in the Wistar rat. Using the (32)P-postlabeling method, three o-anisidine-derived DNA adducts were found in several organs of rats treated with a total dose of 0.53 mg o-anisidine/kg body wt (0.15, 0.18, and 0.2 mg/kg body wt ip in the first, second, and third day, respectively), of which the urinary bladder had the highest levels. At four posttreatment times (1 day, 13 days, 10 weeks, and 36 weeks), DNA adducts in bladder, liver, kidney, and spleen of rats were analyzed to study their persistence. In all time points, the highest total adduct levels were found in urinary bladder (39 adducts per 10(7) nucleotides after 1 day and 15 adducts per 10(7) nucleotides after 36 weeks) where 39% adducts remained. In contrast to the urinary bladder, no persistence was detected in other organs. All three DNA adducts were identified as deoxyguanosine adducts. When deoxyguanosine was reacted with the oxidative metabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine, three adducts could be separated by high-performance liquid chromatography (HPLC) and were identified by mass spectroscopy and/or nuclear magnetic resonance spectrometry. All adducts are products of the nitrenium/carbenium ions, the reactive species generated from N-(2-methoxyphenyl)hydroxylamine. The major adduct was identified to be N-(deoxyguanosin-8-yl)-2-methoxyaniline. Using cochromatography on HPLC, this adduct was found to be identical to the major adduct generated by activation of o-anisidine in vitro and in vivo.

Human cytochrome-P450 enzymes metabolize N-(2-methoxyphenyl)hydroxylamine, a metabolite of the carcinogens o-anisidine and o-nitroanisole, thereby dictating its genotoxicity.

N-(2-Methoxyphenyl)hydroxylamine is a component in the human metabolism of two industrial and environmental pollutants and bladder carcinogens, viz. 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole), and it is responsible for their genotoxicity. Besides its capability to form three deoxyguanosine adducts in DNA, N-(2-methoxyphenyl)-hydroxylamine is also further metabolized by hepatic microsomal enzymes. To investigate its metabolism by human hepatic microsomes and to identify the major microsomal enzymes involved in this process are the aims of this study. N-(2-Methoxyphenyl)hydroxylamine is metabolized by human hepatic microsomes predominantly to o-anisidine, one of the parent carcinogens from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome-b(5) reductase were used to characterize human liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute the main activity for this metabolic step in human liver to CYP3A4, 2E1 and 2C (more than 90%). The enzymes CYP2D6 and 2A6 also partake in this N-(2-methoxyphenyl)hydroxylamine metabolism in human liver, but only to ∼6%. Among the human recombinant CYP enzymes tested in this study, human CYP2E1, followed by CYP3A4, 1A2, 2B6 and 2D6, were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. The results found in this study indicate that genotoxicity of N-(2-methoxyphenyl)hydroxylamine is dictated by its spontaneous decomposition to nitrenium/carbenium ions generating DNA adducts, and by its susceptibility to metabolism by CYP enzymes.

Identification of rat cytochromes P450 metabolizing N-(2-methoxyphenyl)hydroxylamine, a human metabolite of the environmental pollutants and carcinogens o-anisidine and o-nitroanisole.

OBJECTIVES: N-(2-methoxyphenyl)hydroxylamine is a human metabolite of two industrial and environmental pollutants and bladder carcinogens 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole). Metabolism of N-(2-methoxyphenyl)hydroxylamine by rat hepatic microsomes and identification of the major microsomal enzymes participating in this process are aims of this study. METHODS: HPLC with UV detection was employed for the separation of N-(2-methoxyphenyl)hydroxylamine metabolites. Inducers and inhibitors of microsomal enzymes and rat recombinant CYPs were used to characterize the enzymes participating in N-(2-methoxyphenyl)hydroxylamine metabolism. RESULTS: N-(2-methoxyphenyl)hydroxylamine is metabolized by rat hepatic microsomes predominantly to o-anisidine, the parent carcinogen from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome b5 reductase and hepatic microsomes of rats pre-treated with specific inducers of CYPs and NADPH:CYP reductase were used to characterize rat liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute most of N-(2-methoxyphenyl)hydroxylamine metabolism to o-anisidine in rat liver to CYP2C, followed by CYP2E1, 2D and 2A. Among recombinant rat CYP enzymes tested in this study, rat CYP2C11 and 2E1, followed by CYP2A2, 2D1/2, 2C12, 3A1/2 and 1A1/2 were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. CONCLUSION: The results found in this study, the first report on the reduction of N-(2-methoxyphenyl)hydroxylamine by rat CYP enzymes, demonstrate that CYP2C, followed by CYP2E1, 2D and 2A are the major enzymes participating in this process in rat liver.

Genotoxic mechanisms for the carcinogenicity of the environmental pollutants and carcinogens o-anisidine and 2-nitroanisole follow from adducts generated by their metabolite N-(2-methoxyphenyl)-hydroxylamine with deoxyguanosine in DNA.

An aromatic amine, o-anisidine (2-methoxyaniline) and its oxidative counterpart, 2-nitroanisole (2-methoxynitrobenzene), are the industrial and environmental pollutants causing tumors of the urinary bladder in rats and mice. Both carcinogens are activated to the same proximate carcinogenic metabolite, N-(2-methoxyphenyl)hydroxylamine, which spontaneously decomposes to nitrenium and/or carbenium ions responsible for formation of deoxyguanosine adducts in DNA in vitro and in vivo. In other words, generation of N-(2-methoxyphenyl)hydroxylamine is responsible for the genotoxic mechanisms of the o-anisidine and 2-nitroanisole carcinogenicity. Analogous enzymes of human and rat livers are capable of activating these carcinogens. Namely, human and rat cytochorme P4502E1 is the major enzyme oxidizing o-anisidine to N-(2-methoxyphenyl)hydroxylamine, while xanthine oxidase of both species reduces 2-nitroanisole to this metabolite. Likewise, O-demethylation of 2-nitroanisole, which is the detoxication pathway of its metabolism, is also catalyzed by the same human and rat enzyme, cytochorme P450 2E1. The results demonstrate that the rat is a suitable animal model mimicking the fate of both carcinogens in humans and suggest that both compounds are potential carcinogens also for humans.

Isolation and partial characterization of the adduct formed by 13-hydroxyellipticine with deoxyguanosine in DNA.

OBJECTIVES: Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of its action. Recently, we have found that 13-hydroxyellipticine, formed from ellipticine as the predominant metabolite in human livers, is bound to deoxyguanosine in DNA, generating the major DNA adduct in vivo and in vitro. The development of the methods suitable for the preparation of this adduct in the amounts sufficient for identification of its structure and those for its isolation and partial characterization is the aim of this study. METHODS: High performance liquid chromatography (HPLC) was employed for separation of 13-hydroxyellipticine-mediated deoxyguanosine adduct. The 32P-postlabeling technique was utilized to detect this adduct in DNA. RESULTS: The formation of the 13-hydroxyellipticine-derived deoxyguanosine adduct in DNA in vitro was increased under the alkaline pH of the incubations and by the formation of the sulfate and acetate conjugates of 13-hydroxyellipticine generated by reactions with 3'-phosphoadenosine-5'-phosphosulfate (PAPS) or acetyl-coenzyme A (acetyl-CoA) catalyzed by human sulfotransferases (SULTs) 1A1 and 1A2 and N,O-acetyltransferases (NATs) 1 and 2. The HPLC method suitable for separation the 13-hydroxyellipticine-derived deoxyguanosine adduct from other reactants, deoxyguanosine and 13-hydroxyellipticine, was developed. The structure of this adduct is proposed to correspond to the product formed from ellipticine-13-ylium with the exocyclic 2-NH2 group of guanine in DNA. CONCLUSIONS: The data are the first report on HPLC isolation of the deoxyguanosine adduct formed by 13-hydroxyellipticine in DNA and its partial characterization.

Redox cycling in the metabolism of the environmental pollutant and suspected human carcinogen o-anisidine by rat and rabbit hepatic microsomes.

We investigated the ability of hepatic microsomes from rat and rabbit to metabolize 2-methoxyaniline (o-anisidine), an industrial and environmental pollutant and a bladder carcinogen for rodents. Using HPLC combined with electrospray tandem mass spectrometry, we determined that o-anisidine is oxidized by microsomes of both species to N-(2-methoxyphenyl)hydroxylamine, o-aminophenol, and one additional metabolite, the exact structure of which has not been identified as yet. N-(2-Methoxyphenyl)hydroxylamine is either further oxidized to 2-methoxynitrosobenzene (o-nitrosoanisole) or reduced to parental o-anisidine, which can be oxidized again to produce o-aminophenol. To define the role of microsomal cytochromes P450 (P450) in o-anisidine metabolism, we investigated the modulation of this metabolism by specific inducers and selective inhibitors of these enzymes. The results of the studies suggest that o-anisidine is a promiscuous substrate of P450s of rat and rabbit liver; because P450s of 1A, 2B, 2E, and 3A subfamilies metabolize o-anisidine in hepatic microsomes of both studied species. Using purified enzymes of rat and rabbit (P450s 1A1, 1A2, 2B2, 2B4, 2E1, 2C3, 3A1, and 3A6), reconstituted with NADPH:P450 reductase, the ability of P450s 1A1, 1A2, 2B2, 2B4, 2E1, and 3A6 to metabolize o-anisidine was confirmed. In the reconstituted P450 system, rabbit P450 2E1 was the most efficient enzyme metabolizing o-anisidine. The data demonstrate the participation of different rat and rabbit P450s in o-anisidine metabolism and indicate that both experimental animal species might serve as suitable models to mimic the fate of o-anisidine in human.

Cytochrome P450-mediated metabolism of N-(2-methoxyphenyl)-hydroxylamine, a human metabolite of the environmental pollutants and carcinogens o-anisidine and o-nitroanisole.

N-(2-methoxyphenyl)hydroxylamine is a human metabolite of the industrial and environmental pollutants and bladder carcinogens 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole). Here, we investigated the ability of hepatic microsomes from rat and rabbit to metabolize this reactive compound. We found that N-(2-methoxyphenyl)hydroxylamine is metabolized by microsomes of both species mainly to o-aminophenol and a parent carcinogen, o-anisidine, whereas 2-methoxynitrosobenzene (o-nitrosoanisole) is formed as a minor metabolite. Another N-(2-methoxyphenyl)hydroxylamine metabolite, the exact structure of which has not been identified as yet, was generated by hepatic microsomes of rabbits, but its formation by those of rats was negligible. To evaluate the role of rat hepatic microsomal cytochromes P450 (CYP) in N-(2-methoxyphenyl)hydroxylamine metabolism, we investigated the modulation of its metabolism by specific inducers of these enzymes. The results of this study show that rat hepatic CYPs of a 1A subfamily and, to a lesser extent those of a 2B subfamily, catalyze N-(2-methoxyphenyl)hydroxylamine conversion to form both its reductive metabolite, o-anisidine, and o-aminophenol. CYP2E1 is the most efficient enzyme catalyzing conversion of N-(2-methoxyphenyl)hydroxylamine to o-aminophenol.