Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12614–12618, October 1998 Medical Sciences

Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I (replication͞DNA repair͞succinylacetone)

MARIA JOSE´ PRIETO-ALAMO AND FRANC¸OISE LAVAL*

Unite´347 Institut National de la Sante´et de la Recherche Me´dicale,80 Rue du Ge´ne´ralLeclerc, 94276 Le Kremlin Biceˆtre,France

Communicated by Philip Hanawalt, Stanford University, Stanford, CA, July 24, 1998 (received for review December 14, 1997)

ABSTRACT Hereditary tyrosinemia type I (HT1) is an metabolism. FAA is mutagenic in Chinese hamster V79 cells autosomal recessive inborn error of metabolism caused by the (6), and free glutathione concentration in HT1 liver specimens deficiency of fumarylacetoacetate hydrolase, the last enzyme is reduced to about half of normal levels (7). The final in the tyrosine catabolism pathway. This defect results in metabolite, SA, reacts nonenzymatically with proteins and free accumulation of succinylacetone (SA) that reacts with amino amino acids, and the majority of urinary SA in HT1 patients acids and proteins to form stable adducts via Schiff base is found in the form of such adducts (8). formation, lysine being the most reactive amino acid. HT1 As SA reacts with amino acids, and specially with lysine, patients surviving beyond infancy are at considerable risk for generating adducts via Schiff base formation (8), we made the the development of hepatocellular carcinoma, and a high level assumption that SA could react with proteins involved in DNA of chromosomal breakage is observed in HT1 cells, suggesting metabolism and preferentially with proteins whose active site a defect in the processing of DNA. In this paper we show that includes a lysine residue. A lysine residue is present in the the overall DNA-ligase activity is low in HT1 cells (about 20% active site of DNA-ligases that are involved in DNA metabo- of the normal value) and that Okazaki fragments are rejoined lism to rejoin strand interruptions formed transiently during at a reduced rate compared with normal fibroblasts. No replication, repair, and recombination (9). The assumption mutation was found by sequencing the ligase I cDNA from that DNA-ligases could be a target for SA was supported by the HT1 cells, and the level of expression of the ligase I mRNA was observation that fibroblasts from HT1 patients exhibit a high similar in normal and HT1 fibroblasts, suggesting the pres- level of chromosomal breakage (10). ence of a ligase inhibitor. SA was shown to inhibit in vitro the To study whether the accumulation of SA could play a role overall DNA-ligase activity present in normal cell extracts. in the DNA metabolism of HT1 cells, we have measured the The activity of purified T4 DNA-ligase, whose active site is also DNA-ligase activity in fibroblasts from several HT1 patients. a lysine residue, was inhibited by SA in a dose-dependent The influence of SA on the activity of purified T4 DNA-ligase manner. These results suggest that accumulation of SA re- also was investigated, as this enzyme and mammalian ligases duces the overall ligase activity in HT1 cells and indicate that act by an identical mechanism, with the formation of a covalent metabolism errors may play a role in regulating enzymatic lysine–AMP and DNA–AMP reaction intermediates, using activities involved in DNA replication and repair. ATP as the source of AMP (11).

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease that has been reported worldwide but with variable MATERIALS AND METHODS

frequency. Two forms of the disease have been described. The Chemicals. Poly(A) and oligo(dT)16 were from Pharmacia. acute form is characterized by liver failure, and patients usually [␣-32P]ATP and [␥-32P]ATP (3,000 Ci͞mmol; 1 Ci ϭ 37 GBq) die within the first year of life. In the chronic form, patients were obtained from Amersham. Succinylacetone (Sigma) was show renal tubular dysfunction, hypophosphatemic rickets, dissolved at a concentration of 500 mM in 1 M Hepes, pH 7.5, progressive liver disease, and a high incidence of hepatocel- then diluted to the appropriate concentrations in the incuba- lular carcinoma (1). tion buffers. T4 DNA-ligase was from Boehringer Mannheim. It has been shown that HT1 is caused by a deficiency of Cell Culture. Fibroblasts from five unrelated HT1 patients fumarylacetoacetate hydrolase (FAH), the enzyme involved in were obtained from skin biopsies. One of these HT1 cell lines the last step of the catabolic pathway of tyrosine (2). The was designated as ALV fibroblasts. Normal (MRC 5) and HT1 human cDNA encoding FAH has been cloned and the FAH fibroblasts were maintained in Ham’s F12 (GIBCO) supple- gene has been located to region q23–q25 of chromosome 15 mented with 10% fetal calf serum and 5% horse serum, (3). The gene contains 14 exons and spans approximately 35 kb penicillin, and streptomycin. The doubling times were about 30 of DNA (4). The enzyme is expressed predominantly in the and 50 hr for normal and HT1 fibroblasts, respectively. LICH liver but is also found in a wide range of tissues and cell types cells (derived from a human hepatoma) (12) were grown in (5). The FAH deficiency results in an accumulation of two Dulbecco’s medium supplemented with 5% fetal calf serum abnormal tyrosine metabolites, fumarylacetoacetate (FAA) and 5% horse serum in the presence of antibiotics. and maleylacetoacetate (MAA), which, after reduction and To prepare cell extracts, exponentially growing cells were decarboxylation, are transformed to succinylacetone (SA). SA harvested by trypsinization, washed in Earle’s balanced salt is excreted in the urine and its finding is part of the HT1 solution (EBSS), and suspended (5 ϫ 107 cells per ml) in a diagnosis. It has been suggested that FAA and MAA possibly buffer containing 66 mM Hepes at pH 7.5, 5 mM MgCl ,1 mM act as natural alkylating agents and͞or disrupt sulfhydryl 2 DTT, and 10% glycerol. After addition of protease inhibitors,

The publication costs of this article were defrayed in part by page charge Abbreviations: HT1, hereditary tyrosinemia type I; SA, succinylac- payment. This article must therefore be hereby marked ‘‘advertisement’’ in etone; FAH, fumarylacetoacetate hydrolase; FAA, fumarylacetoac- accordance with 18 U.S.C. §1734 solely to indicate this fact. etate; MAA, maleylacetoacetate. © 1998 by The National Academy of Sciences 0027-8424͞98͞9512614-5$2.00͞0 *To whom reprint requests should be addressed. e-mail: laval@kb. PNAS is available online at www.pnas.org. inserm.fr.

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cells were disrupted by sonication at 0°C, and cell debris was The overall DNA-ligase activity was measured by the joining ⅐ removed by centrifugation (13). of single-strand interruptions in the oligo(dT)16 poly(dA) sub- Reverse Transcription, PCR, and Sequencing. Five micro- strate, in extracts from ALV (Fig. 1A) and normal human grams of total RNA, isolated from normal or HT1 fibroblasts fibroblasts (MRC5 cells) (Fig. 1B). In the same experimental (14), was reverse-transcribed by using the First-strand cDNA conditions, the kinetics experiments showed a low activity in synthesis kit (Boehringer Mannheim) according to the instruc- ALV fibroblasts, reduced about 5-fold in comparison with the tions of the manufacturer. The FAH and ligase I cDNA were activity present in the normal fibroblasts extracts (Fig. 1C). amplified by PCR in a DNA thermal cycler (Perkin–Elmer͞ Similar results were obtained when the DNA-ligase activity Cetus) by using the PCR Expand kit (Boehringer) and specific was measured in fibroblasts from five unrelated HT1 patients primers (15). They were sequenced by using the Applied and compared with that present in normal human fibroblasts Biosystems (ABI Prism 310 Genetic Analyzer) Taq DyeDeoxy (MRC5 cells) and human hepatoma cells (LICH cells) (Fig. 2). Terminator Cycle Sequencing procedure, following the man- In the five HT1 cell lines, the activity was low and corre- ufacturer’s specifications. To determine the amount of ligase sponded to about 20–30% of the level measured in non-HT1 I mRNA, increasing amounts of total RNA were reverse- cells. transcribed and then amplified by using specific primers. PCR To check that this low DNA-ligase activity was not a result of an unexpected lability of the enzymes present in HT1 cells conditions were determined to obtain an exponential ampli- fication. The PCR products were run on a 1.8% agarose gel and quantified. Formation of DNA-Ligase AMP Intermediates. T4 DNA- ligase (2 units, 0.150 ␮g of proteins) was incubated (final volume, 10 ␮l) in a buffer containing 66 mM Hepes at pH 8.0, 10 mM MgCl2, and increasing SA concentrations for 15 min at 25°C. The protein was incubated further for 15 min at 25°C after addition of 10 ␮l of the same buffer containing [␣-32P]ATP diluted with nonradioactive ATP to a specific activity of 250 Ci͞mmol and added to a final concentration of 2 ␮M ATP (16). The reaction products were analyzed by electrophoresis in SDS͞polyacrylamide gels and quantified by densitometry. DNA-Ligase Activity Determination. The substrate was [5Ј- 32 ⅐ P]oligo(dT)16 poly(dA), prepared as described (17). The substrate (0.25 ␮g, about 20,000 cpm) was incubated in 66 mM ͞ ͞ ␮ Hepes, pH 7.5 5 mM MgCl2 35 M ATP, with the T4 DNA-ligase (0.5 unit) in a final volume of 20 ␮l. Samples were incubated at 37°C for 5 min. The reaction was stopped by the addition of formamide (10 ␮l) and heating at 100°C for 5 min. The reaction products were separated in 15% polyacryl- amide͞7 M urea gels and quantified by using an InstantImager (Packard). In the case of cellular extracts, incubation was for 30 min at 37°C, and then the samples were treated with an equal volume of phenol͞chloroform and centrifuged, and the aqueous phase was analyzed by electrophoresis. Analysis of Okazaki Fragments. Cells were synchronized by isoleucine deprivation for 48 hr followed by release in com- plete medium containing aphidicolin (10 ␮g͞ml) for 12 hr, washed, and cultured in normal medium to transit to S phase. They were collected, suspended in replication mixture, per- meabilized by addition of Nonidet P-40, and incubated in the presence of [␣-32P]dATP as described (16). DNA replication was allowed for 1 min, and then the reaction was chased for increasing periods of time by adding nonradioactive dATP (10 mM final concentration). Samples were treated as described (16) and analyzed by electrophoresis in 10% polyacrylamide͞8 M urea gels, and the formation of high molecular DNA was quantified by densitometry.

RESULTS DNA-Ligase Activity in HT1 Fibroblasts. To confirm the clinical diagnosis of tyrosinemia type I in the ALV fibroblasts used in this study, the FAH cDNA (1,396 bp) was amplified by reverse transcription–PCR and sequenced. Two patterns were FIG. 1. DNA-joining activity in ALV fibroblasts. Extracts (25 ␮g superposed for exon 12 of the ALV cDNA. One pattern of proteins) from ALV (A) or normal (B) fibroblasts were incubated Ј 32 ⅐ contained a C-to-G transversion changing serine to glycine in with the polynucleotide substrate [5 - P]oligo(dT)16 poly(dA) at 37°C codon 348. The second pattern showed a deletion of the first for 0 (lane 1), 15 (lane 2), 30 (lane 3), 60 (lane 4), 90 (lane 5), or 120 min (lane 6). The oligo(dT)16 multimers were separated in polyacryl- 50 bp of exon 12. This deletion was confirmed by the loss of the amide͞urea gels. The films were overexposed to detect the small PstI site located in exon 12 (data not shown). These results amounts of multimers in A.(C) The data from A and B are expressed characterize the FAH defect in ALV cells and confirm the in femtomoles of oligo(dT)16 ligated. (F) ALV and (Œ) normal clinical diagnosis of tyrosinemia type I. fibroblasts. Downloaded by guest on September 24, 2021 12616 Medical Sciences: Prieto-Alamo and Laval Proc. Natl. Acad. Sci. USA 95 (1998)

FIG. 2. DNA-ligase activity in fibroblasts from various HT1 pa- tients. Extracts (25 ␮g of proteins) from normal fibroblasts (lane 1), LICH cells (lane 2), or fibroblasts from five unrelated HT1 patients (lanes 3–7) were incubated with the polynucleotide substrate [5Ј- 32 ⅐ P]oligo(dT)16 poly(dA) for 120 min at 37°C. The oligo(dT)16 mul- FIG. 3. Joining of Okazaki fragments in normal and ALV fibro- ͞ timers were detected by electrophoresis in polyacrylamide urea gels blasts. Cells synchronized in S phase were incubated in the presence and autoradiography. For details, see Materials and Methods. of [␣-32P]dATP for 1 min. The reaction then was chased by incubation with nonradioactive dATP for different times. After DNA isolation, extracts, two other activities were measured. The glyceralde- replication intermediates were analyzed by gel electrophoresis and hyde-3-phosphate dehydrogenase (18) and the N3-methylad- detected by autoradiography. The amount of low-molecular-weight enine DNA glycosylase (19) had identical activities in HT1, DNA corresponding to Okazaki fragments was estimated by densi- normal fibroblasts, and human hepatoma cells (LICH cells) tometry. (Œ) Normal and (F) ALV fibroblasts. (data not shown). Joining of Okazaki Fragments in HT1 Cells. To investigate inhibitor was SA, which accumulates in HT1 cells and forms whether this low overall ligase activity could impair DNA lysine adducts. To test this hypothesis, the influence of this replication in HT1 cells, synthesis and joining of Okazaki compound was measured, either on the activity of purified T4 fragments were measured. Normal and ALV cells were syn- DNA-ligase or on the activity present in normal cell extracts. ͞ T4 DNA-ligase was incubated for 15 min at 25°C with chronized at the G1 S boundary and pulse-labeled with [␣-32P]dATP, and the newly synthesized DNA fragments were increasing SA concentrations, and its activity was measured by chased with nonradioactive dATP for different time lengths. the joining of single-strand interruptions in the double- Ј 32 ⅐ There was a slight difference in the conversion of the frag- stranded [5 - P]oligo(dT)16 poly(dA) substrate. As shown by ments to high-molecular-weight DNA after a 2-min chase analysis in polyacrylamide gels, the T4 DNA-ligase activity period, but about 35% of the DNA remained in low molecular decreases after incubation with SA (Fig. 4A). This decrease is ␮ weight in ALV cells during the period studied (20 min), dose-dependent with an IC50 of about 15 M (Fig. 4B). When ␮ whereas no such fragments were observed in normal cells (Fig. the protein was incubated with SA (25 M) in the presence of ␮ 3). free lysine (25 M), 70% of the activity was recovered. This It should be noted that, in our experimental conditions, the significant protection of the activity suggests that the inhibition doubling time for ALV fibroblasts (about 50 hr) is longer than is due to the reaction of SA with the lysine residue within the that of normal cells (30 hr) and this could be a consequence active site of the protein. Increasing the incubation times with of the impaired DNA replication. SA did not enhance further the inhibitory effect of this Sequencing and Expression of the Ligase I cDNA from ALV compound on the enzyme activity (data not shown). The first Fibroblasts. DNA-ligase I represents an important part of the step of the ligation reaction is the formation of a covalent total activity present in human cells and is involved primarily ligase–AMP intermediate with a lysine–adenylate phospho- in DNA replication. The low overall ligase activity and the amide bond. The influence of SA on this step of the reaction reduced rate of rejoining of Okazaki fragments suggested that was measured by incubating the T4 DNA-ligase for 15 min at ligase I activity was defective in ALV cells. Therefore, the 25°C with increasing SA concentrations before incubation with ligase I cDNA from ALV fibroblasts was sequenced. The [␣-32P]ATP. The formation of the DNA-ligase adenylate results showed the absence of mutations in this gene. Further- complex is inhibited in the presence of SA (Fig. 4C). The more, the level of expression of ligase I mRNA, measured by amount of adenylylated protein decreases with the SA con- ␮ reverse transcription–PCR, was identical in normal and ALV centration with an IC50 of about 20 M, showing that the fibroblasts (data not shown). inhibition occurs at the first step of the ligation reaction, the Influence of Succinylacetone on the T4 DNA-Ligase Activ- enzyme–AMP formation. For comparison, the protein was ity. The results described above suggested the presence of a incubated in the same conditions with pyridoxal phosphate DNA-ligase inhibitor in ALV cells. An inhibitor, which is a that is known to interact with lysine residues and to inhibit the heat-labile protein, has been described in extracts from human DNA-ligase activity (21). The formation of T4 DNA-ligase cells (20). To test whether the low activity measured in ALV adenylate was inhibited by 70% in the presence of 50 ␮M cells was a result of the presence of this protein, extracts from pyridoxal 5Ј-phosphate (not shown). ALV cells were heated to 65°C for 30 min and then added to Incubating normal fibroblast extracts (25 ␮g of proteins) normal cell extracts. The activity of normal cell extracts was with SA (100 ␮M) for 15 min reduced the overall ligase activity decreased by 40% when ALV extracts, heated or not, were to 50% of the control value (Fig. 5). This shows that SA is able added to the incubation medium, showing that these extracts to inhibit the ligase activity even in presence of the large contain an inhibitor that is not a heat-labile protein. A possible amount of proteins contained in this crude extract. Downloaded by guest on September 24, 2021 Medical Sciences: Prieto-Alamo and Laval Proc. Natl. Acad. Sci. USA 95 (1998) 12617

FIG. 5. Influence of SA on the DNA-ligase activity present in normal cell extracts. Normal fibroblasts extracts (25 ␮g proteins) were preincubated for 15 min at 25°C without (Œ)orwith(F) SA (100 ␮M). The ligase activity was then measured as described in Materials and Methods.

anedione treatment (28), which blocks the accumulation of metabolites upstream of FAH, are, to date, the only therapies for this disease. In the chronic form of HT1, when patients survive beyond 2 years of age, there is a high incidence (37%) of hepatocellular carcinoma, which is an uncommon tumor in children (29). DNA damage occurs by spontaneous base deamination, alkylation, or oxidation by endogenous or environmental exposure to various compounds. Repair of these damages and correction of replicative errors are critical to maintain the genome integrity, and DNA-repair deficiency syndromes have an increased risk of developing cancer (reviewed in ref. 30). Therefore, the high cancer incidence occurring in HT1 pa- tients, the high level of chromatid breaks observed in HT1 cells (10), and the hypersensitivity of established HT1 cells to several DNA-damaging agents (F.L., unpublished results) lead to the hypothesis that enzymes involved in DNA repair and͞or replication could be altered in HT1 cells. Different DNA-ligases have been described in mammalian cells (31, 32) and are implicated in DNA replication and in recombination and are required in the final step of base FIG. 4. T4 DNA-ligase activity in the presence of SA. T4 DNA- excision and nucleotide excision repair (33). As diketones react ligase was treated or not with increasing SA concentrations (15 min at with the ␧-amino group of lysine (34), among the possible 25°C) and then incubated at 37°C for 1 min with the oligo substrate. targets for the formation of a Schiff base with SA was the lysine ͞ (A) The oligo(dT)16 multimers were separated in polyacrylamide urea residue present in the active site of mammalian ligases. Our gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing results show that the overall DNA-ligase activity measured in SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) ␮M. (B) The activity was quantitated using an InstantImager (Packard). (C) Inhi- cells from unrelated HT1 patients corresponds to about 20% bition of enzyme-adenylate formation by SA. T4 DNA-ligase was of the activity present in normal human fibroblasts. SA reduces incubated or not with increasing SA concentrations (15 min at 25°C) the activity present in normal cells extracts as well as the before the addition of [␣-32P]ATP. The enzyme adenylate complexes activity of purified T4 DNA-ligase, whose active site is also a were separated by electrophoresis and detected by autoradiography. lysine residue, strongly suggesting that this compound is re- sponsible for the low activity measured in HT1 cells. This DISCUSSION deficiency results in a slow rejoining of Okazaki fragments in HT1 cells, probably plays a role in the completion of DNA Tyrosinemia type I is caused by a deficiency of FAH, and repair processes, and might result in genomic instability. It has different mutations have been identified in the FAH gene, been suggested that the extent of DNA-ligase I deficiency including missense, nonsense, and splice consensus site mu- tolerable to mammalian cells is low and that ligases I, II, and tations (15, 22–24). However, there is no strict correlation III are unable to compensate for each other (35, 36). Hence, between genotype and phenotype, i.e., the acute, subacute, or the overall ligase deficiency observed in HT1 cells probably chronic form of the disease (24). The lack of FAH results in plays a key role in the symptoms associated with this disease. the formation of FAA and MAA, but these two compounds A human cell line (46BR) is sensitive to killing by DNA- have never been isolated as circulating or excreted metabolites damaging agents (37), shows defective rejoining of Okazaki (25). This suggests that they are transformed rapidly to SA, and fragments, and possesses reduced DNA-ligase I activity (16). SA concentrations from 6 to 43 ␮M were measured in the Sequencing of PCR-amplified ligase I cDNA revealed muta- plasma of HT1 patients (26). Attempts have been made in vitro tions in 46BR cells, carried in different alleles (38). As to express the FAH cDNA in cultured HT1 cells by using contrasted to 46BR cells, no mutation was detected in the retroviral-mediated gene transfer (27), but liver transplanta- ligase I cDNA of HT1 cells, and the level of transcription of this tion and 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohex- gene was identical in normal and HT1 cells. Although this Downloaded by guest on September 24, 2021 12618 Medical Sciences: Prieto-Alamo and Laval Proc. Natl. Acad. Sci. USA 95 (1998)

activity is reduced in both 46BR and HT1 cells, the clinical 14. Chirgwin, J., Przybyla, A., MacDonald, R. & Rutter W. J. (1979) symptoms associated with the diseases are different (38), Biochemistry 18, 5294–5299. suggesting that besides its inhibitory effect on DNA-ligases, SA 15. Rootwelt, H., Chou, J., Gahl, W. A., Berger, R., Coskun, T., may have other deleterious effects on the cellular metabolism. Brodtkorb, E. & Kvittingen, E. A. (1994) Hum. Genet. 93, 615–619. The precise influence of SA on the different mammalian 16. Prigent, C., Satoh, M. S., Daly, G., Barnes, D. E. & Lindahl, T. DNA-ligases now should be determined to correlate the level (1994) Mol. Cell. Biol. 14, 310–317. of each activity with specific defects observed in HT1 cells. The 17. Yang, S. W. & Chan, J. Y. H. (1992) J. Biol. Chem. 267, influence of SA on other enzymes also has to be tested, 8117–8122. especially on enzymes whose active site includes a lysine 18. Sirover, M. A. (1997) J. Cell. 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