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Mutational analyses of the thermostable NAD+-dependent DNA ligase from Thermus filiformis
FEMS Microbiology Letters 237 (2004) 111–118 www.fems-microbiology.org
Mutational analyses of the thermostable NADþ-dependent DNA ligase from Thermus filiformis Hyo Jeong Jeon a, Hea-Jin Shin a, Jeong Jin Choi a, Hyang-Sook Hoe a, Hyun-Kyu Kim b, Se Won Suh c, Suk-Tae Kwon a,* a
Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea b Genetic Resource R&D Institute, Super Bio Co., Ltd., Suwon 440-746, Republic of Korea c Department of Chemistry, Seoul National University, Seoul 151-742, Republic of Korea Received 17 February 2004; received in revised form 19 May 2004; accepted 14 June 2004 First published online 22 June 2004
Abstract The crystal structure of NADþ -dependent DNA ligase from Thermus filiformis (Tfi) revealed that the protein comprised four structural domains. In order to investigate the biochemical activities of these domains, seven deletion mutants were constructed from the Tfi DNA ligase. The mutants Tfi-M1 (residues 1–581), Tfi-M2 (residues 1–448), Tfi-M3 (residues 1–403) and Tfi-M4 (residues 1– 314) showed the same adenylation activity as that of wild-type. This result indicates that only the adenylation domain (domain 1) is essential for the formation of enzyme–AMP complex. It was found that the zinc finger and helix-hairpin-helix (HhH) motif domain (domain 3) and the oligomer binding (OB)-fold domain (domain 2) are important for the formation of enzyme–DNA complex. The mutant Tfi-M1 alone showed the activities for in vitro nick-closing and in vivo complementation in Escherichia coli as those of wildtype. These results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the enzyme activity. The enzymatic properties of Tfi-M1 mutant (deleted the BRCT domain) were slightly different from those of wild-type and the nickclosing activity of Tfi-M1 mutant was approximately 50% compared with that of wild-type. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Thermus filiformis (Tfi) DNA ligase; Self-adenylation; DNA binding activity; Nick-closing activity
1. Introduction DNA ligases (EC 6.5.1.1 and EC 6.5.1.2) catalyze the sealing of 50 -phosphate and 30 -hydroxyl termini at single-strand breaks in double-stranded DNA, or at two fragments containing either complementary single strand or blunt ends, which are essential in DNA replication, recombination and repair [1]. Early studies on Escherichia coli and T4 DNA ligases have demonstrated the different cofactor requirements and elucidated the basic enzymatic mechanism of DNA ligation. ATP-
*
Corresponding author. Tel.: +82-31-290-7863; fax: +82-31-2907870. E-mail address: [email protected] (S.-T. Kwon).
dependent DNA ligases have been found in bacteriophages, archaea, eukaryotes, and recently in bacteria, whereas NADþ -dependent DNA ligases have exclusively been found in bacteria [2]. Apart from the difference in cofactor requirement, the reactions catalyzed by the two classes of DNA ligase are identical. In the first step, an AMP group derived from either cofactor is covalently attached to the conserved lysine residue within the Lys–Xaa–Asp–Gly (KXDG) motif. The AMP moiety is then transferred from the adenylated enzyme intermediate to the free 50 -phosphoryl group at a nicked site of duplex DNA. Finally, the AMP group is released from the adenylated DNA intermediate as the phosphodiester bond is formed [1]. Bacterial ligases from thermophilic organisms have become model systems for structural and mechanistic
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.06.018
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studies of DNA ligation due to their high thermostability. Recently, the thermostable DNA ligase is the key component of the ligase chain reaction (LCR) for amplifying and detecting a single-base genetic disease [3]. Studies on the properties of Thermus thermophilus HB8 (Tth) DNA ligase have been first reported among thermostable DNA ligases [4,5]. This enzyme is active at temperature above 70 °C. The genes encoding Tth DNA ligase [6], T. scotoductus DNA ligase [7] and T. filiformis (Tfi) DNA ligase [8] were cloned into E. coli, respectively, and their nucleotide sequences and deduced amino acid sequences were reported. Especially, Tfi DNA ligase gene contains an open reading frame encoding 667 amino acids with a molecular weight of 75,936 Da [8]. The crystal structure of Tfi DNA ligase, a 667-residue multidomain protein, has recently been solved [9]. Tfi DNA ligase was found to have a unique circular arrangement of its four distinct domains, domain 1 (residues 1–317, adenylation domain), domain 2 (residues 318–403, oligomer binding (OB)-fold domain), domain 3 (residues 404–581, zinc finger and helix-hairpin-helix (HhH) motif domain) and domain 4 (residues 582–667, BRCT domain), which leads to a hole large enough to hold a double-strand DNA. In this paper, we report the cloning and the purification of seven deletion mutants of Tfi DNA ligase, and the biochemical characteristics of the deletion mutants, to define the essential domain(s) for self-adenylation, DNA binding activity and nick-closing activity of the NADþ -dependent Tfi DNA ligase.
2. Materials and methods 2.1. Bacterial strains E. coli BL26Blue {ompT hsdSB (rB mB ) gal dcm lac [F0 proAB lacIq Z DM15:: Tn10 (TcR )]} was used as the host for plasmid preparations and gene expression. E. coli GE1720 (provided by Dr. Sigrıdur H. Thorbjarnard ottir, University of Iceland) was used as the host for in vivo complementation test of DNA ligase activity. This strain carries the ligts251 mutation which had previously been shown to cause loss of viability at 41 °C due to inability to perform ligation of Okazaki fragments [10]. 2.2. Cloning of the deletion mutants of Tfi DNA ligase Standard procedures for DNA ligation, transformation and other cloning-related techniques were as described by Sambrook et al. [11]. The oligonucleotides used as primers were synthesized to clone deletion mutants based on the nucleotide sequence of Tfi DNA ligase gene [8] and the crystal structure of Tfi DNA ligase [9]. The sense primers added a unique EcoRI site
(underlined), and the antisense primers added a unique SalI site (underlined). Recombinant plasmids pTFM1, pTFM2, pTFM3 and pTFM4, which express Tfi DNA ligases deleting 86, 219, 264 and 353 amino acid residues from the C-terminus (designate as Tfi-M1 (residues 1– 581), Tfi-M2 (residues 1–448), Tfi-M3 (residues 1–403) and Tfi-M4 (residues 1–314), respectively), were constructed by a method similar to that described previously [8]. PCR was done with pTFL (contains the Tfi DNA ligase gene)[8] as a template using a common sense primer of 50 -AGACTGAATTCATGACCCGGGAAGA-30 (N-terminal region) and four different antisense primers, that is, 50 -NNNNGTCGACTCACTTGGACTCCATGCTCAC-30 (C-terminal region of Tfi-M1), 50 -NNNNGTCGACTCAGCCCTCTATGTCCATG-30 (C-terminal region of Tfi-M2), 50 -NNNNGTCGACTCAGGGCCAGCGGATGGGCCG-30 (C-terminal region of Tfi-M3), and 50 -NNNNGTCGACTCAGGGGAACTTGTAGGCGAG-30 (Cterminal region of Tfi-M4). Recombinant plasmids pTFM5, pTFM6 and pTFM7, which express Tfi DNA ligases deleting 314, 403 and 581 amino acid residues from the N-terminus (designate as Tfi-M5 (residues 315– 667), Tfi-M6 (residues 404–667) and Tfi-M7 (residues 582–667), respectively), were also constructed. PCR was done with pTFL [8] as a template using a common antisense primer of 50 -CTGGGGTCGACTCAGGCCGGGACGG-30 (C-terminal region) and three different sense primers, that is, 50 -NNNNGAATTCATGGCCGAGGAGAAGGAGACC-30 (N-terminal region of Tfi-M5), 50 -NNNNGAATTCATGGAGGCCTGTCC CGAGTGC-30 (N-terminal region of Tfi-M6), and 50 -NNNNGAATTCATGGAGGAGGTCTCGGAC-30 (N-terminal region of Tfi-M7). Each of the PCR products was digested with EcoRI and SalI, and ligated into the expression vector pJR [12] that had been digested with the same enzymes, giving a fusion that used the tac promoter. E. coli BL26Blue was transformed with the ligates, respectively. Each of the all seven recombinant plasmids was identified by screening a transformants.
2.3. Purification of wild-type and mutants of Tfi DNA ligase expressed in E. coli Ten ml of an overnight culture of E. coli BL26Blue harbouring the recombinant plasmid grown in an Lbroth containing ampicillin were transferred to 1 l of the same medium and cultured at 37 °C, respectively. When the A600 of the culture was about 0.8, the culture was induced by the addition of isopropyl-b-D -thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM, and then incubated at 37 °C for another 6 h. The cells were collected by centrifugation, resuspended in 20 ml of sonication buffer (10 mM Tris–HCl, pH 7.6, and 2 mM MgCl2 ) containing 1 mM phenylmethylsulfonyl fluoride
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(PMSF), then disrupted by sonication. The disrupted cells were centrifuged at 10,000g, 4 °C for 20 min to remove E. coli cell debris. The sonicated extract was incubated at 80 °C for 30 min. The nucleic acids in the supernatant were precipitated by the addition of 1% streptomycin sulfate at room temperature with stirring for 30 min, then the precipitate was removed by centrifugation. The supernatant was dialyzed against buffer A (10 mM Tris–HCl, pH 7.6, 1 mM EDTA and 1 mM PMSF), then applied onto a DEAE–Sephacel column (1.5 11.3 cm) that had been equilibrated with buffer A. The adsorbed protein was eluted with a linear gradient of KCl (0–0.5 M) prepared in 120 ml of buffer A. The polypeptide composition of the column fractions was monitored by SDS–PAGE [13] on a 10% polyacrylamide gel and the DNA ligase fractions were pooled and dialyzed. Protein concentration was determined by the procedure of Lowry et al. [14] with bovine serum albumin (BSA) as a standard. 2.4. Adenylation assay The adenylation reactions were performed by incubating 100 pmol of Tfi DNA ligase and deletion mutants with 1 lCi [32 P]NADþ (800 Ci/mmol, Amersham Biosciences, UK) in a 10 ll reaction mixture (20 mM Tris– HCl, pH 8.0, 10 mM KCl, 5 mM MgCl2 and 2 mM DTT) at 70 °C for 10 min. The reaction was stopped by boiling in SDS loading buffer for 5 min and was analyzed by electrophoresis on a 10% SDS–polyacrylamide gel. The gel was dried, and the adenylated protein bands were analyzed by autoradiography. 2.5. Ligase substrate The substrate used in DNA binding and ligation assays was a 50 bp DNA duplex containing a centrally placed nick. The DNA duplex was formed by annealing two 25mer oligodeoxyribonucleotides to a complementary 50mer template strand as follows. The 3MP18 oligomer (50 -ATCCCCGGGTACCGAGCTCGAATTC-30 , 50 pmol) complementary to the 50 side of the template was radiolabeled by T4 polynucleotide kinase in the presence of 200 lCi [c-32 P]ATP (3000 Ci/mmol, Amersham Biosciences, UK) at 37 °C for 1 h, and purified by ethanol precipitation with ammonium acetate. The labeled oligomer was annealed to the template strand (mp18-Tem, 50 -GAATTCGAGCTCGGTACCCGGG GATCCTCTAGAGTCGACCTGCAGGCATG-30 ) in the presence of the 5MP18 oligomer (50 -CATGCCTGCAGGTCGACTCTAGAGG-30 ), com- plementary to the 30 side of the template, in annealing buffer (20 mM Tris–HCl, pH 8.0, and 10 mM KCl). The mixture was slowly cooled to room temperature after heating at 95 °C for 5 min. The molar ratio of the components,
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3MP18, 5MP18 and mp18-Tem in annealing mixture was 1:1.2:1.2, respectively. 2.6. DNA binding and ligation assays For DNA binding assay, reaction mixtures (10 ll) containing 20 mM Tris–HCl, pH 8.0, 10 mM KCl, 2 mM DTT, 0.2 pmol of 32 P-labeled nicked duplex substrate and 100 pmol of Tfi DNA ligase and deletion mutants were incubated at 50 °C for 30 min [15,16]. The reacted samples were electrophoresed through a native 10% polyacrylamide gel in TBE (90 mM Tris–borate and 2.5 mM EDTA) at 70 V for 2 h. Ligase–DNA complexes were visualized by autoradiography of the dried gel. For DNA ligation assay, reaction mixtures (10 ll) containing 20 mM Tris–HCl, pH 8.0, 10 mM KCl, 5 mM MgCl2 , 2 mM DTT, 0.5 mM NADþ , 0.2 pmol of 32 P-labeled nicked duplex substrate and 100 pmol of Tfi DNA ligase and deletion mutants were incubated at 60 °C for 1 h [15,16]. After incubation, reactions were halted by addition of EDTA and formamide. The reacted samples were heated at 95 °C for 5 min, and then electrophoresed through a 15% polyacrylamide gel containing 7 M urea in TBE. The ligation products were visualized by autoradiography of the dried gel. 2.7. Nick-closing activity assay The assay for nick-closing activity of DNA ligase was performed by the method of Thorbjarnard ottir et al. [17]. The sequences of oligodeoxyribonucleotides used in substrate preparation for nick-closing activity assay were the same as described above; however, the 3MP18 oligomer was labeled with biotin at 30 -end and phosphorylated at 50 -end, and the 5MP18 oligomer was labeled with fluorescein at 50 -end. Tfi DNA ligase and deletion mutants (0.2 pmol) were mixed with 5 pmol of the annealed substrate in 20 ll of standard assay buffer (20 mM Tris–HCl, pH 8.0, 10 mM KCl, 5 mM MgCl2 , 0.5 mM NADþ and 0.01% BSA) and overlayed with paraffin oil. The DNA ligase activity was assayed at 70 °C for 5 min. The reactions were terminated by the addition of 2 ll of 0.1 M EDTA and incubated on ice for 10 min. Triplicate aliquots of 5 ll from each reacted sample were then applied to wells in streptavidin coated 96-well microtiter plate (MaxiSorp; Nunc, Denmark) with 45 ll of washing buffer (100 mM Tris–HCl, pH 8.0, 150 mM NaCl and 0.05% Tween20). The plate was incubated at 37 °C for 1 h, washed once with sterilized water, twice with denaturation buffer (0.1 N NaOH and 0.05% Tween20), twice with sterilized water, once more with washing buffer, and incubated at 37 °C for 30 min with 50 ll of a 1:3000 diluted anti-fluorescein-alkaline phosphatase conjugate (Boehringer-Mannheim, Germany) in washing buffer. After washing six times with
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washing buffer, 100 ll of fresh substrate solution (5 mM p-nitrophenyl phosphate, 100 mM Tris–HCl, pH 9.0, 1 mM MgCl2 and 0.5 mM ZnCl2 ) were added and incubated at 37 °C for 10 min. The resulting yellow color was read by a microplate reader at 405 nm.
3. Results and discussion 3.1. Cloning and purification of the deletion mutants of Tfi DNA ligase A total of seven deletion mutants were constructed based on the X-ray crystal structure of Tfi DNA ligase [9] (Fig. 1). To define the function of the multidomains, four C-terminal deletion mutants, Tfi-M1 (residues 1– 581), Tfi-M2 (residues 1–448), Tfi-M3 (residues 1–403) and Tfi-M4 (residues 1–314), and three N-terminal deletion mutants, Tfi-M5 (residues 315–667), Tfi-M6 (residues 404–667) and Tfi-M7 (residues 582–667), were constructed. The wild-type Tfi DNA ligase and deletion mutants were expressed in E. coli BL26Blue as soluble proteins. The expression level was approximately 5–9% of the total E. coli protein for the wild-type Tfi DNA ligase and deletion mutants. The expressed cells were harvested and ultrasonicated. The proteins were then purified by heat treatment (to denature the E. coli proteins) and one step of DEAE–Sephacel column chromatography. The purified proteins were above 95% pure as judged by densitometric evaluation of proteins separated by SDS–PAGE (Fig. 2). 3.2. Analysis of the enzyme-AMP formation The adenylation of wild-type Tfi DNA ligase and seven deletion mutants were compared with one an-
Fig. 2. SDS–PAGE analysis of wild-type Tfi DNA ligase and deletion mutants. The purified proteins were analyzed on 10% SDS–polyacrylamide gel and stained with Coomassie brilliant blue R-250. Lane 1, wild-type Tfi DNA ligase; lane 2, Tfi-M1 mutant (residues 1–581); lane 3, Tfi-M2 mutant (residues 1–448); lane 4, Tfi-M3 mutant (residues 1–403); lane 5, Tfi-M4 mutant (residues 1–314); lane 6, Tfi-M5 mutant (residues 315–667); lane 7, Tfi-M6 mutant (residues 404–667); lane 8, Tfi-M7 mutant (residues 582–667); lane M, low-molecularweight markers (molecular masses are indicated at the right).
other by incubating each type of DNA ligase in the presence of [32 P]NADþ , followed by SDS–PAGE (Fig. 3(a)). It was shown that the wild-type and four Cterminal deletion mutants formed enzyme–AMP complexes. This result indicates that the N-terminal adenylation domain (domain 1) is essential for the formation of enzyme–AMP complex. The conserved lysine residue within the Lys–Xaa–Asp–Gly motif has been reported as the adenylation residue for the selfadenylation of DNA ligases [15,18]. We also proposed that the adenylation site of Tfi DNA ligase is the Lys116 of Lys–Val–Asp–Gly motif [8,9]. Recently, it has been reported that domain Ia within the adenylation
Fig. 1. Schematic representation of the deletion mutants of Tfi DNA ligase. Domains are in different sizes [9]: domain 1 (residues 1–317, adenylation domain), domain 2 (residues 318–403, OB-fold domain), domain 3 (residues 404–581, zinc finger and HhH motif domain) and domain 4 (residues 582–667, BRCT domain). Solid bars indicate the wild-type (Tfi-WT) and seven deletion mutants (Tfi-M1, M2, M3, M4, M5, M6 and M7) of Tfi DNA ligase.
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philus [16] NADþ -dependent DNA ligases composed solely of the adenylation domain retain full adenylation activity, unlike ATP-dependent DNA ligases. 3.3. Analysis of DNA binding
Fig. 3. Biochemical activities of wild-type Tfi DNA ligase and deletion mutants. (a) Adenylation of wild-type Tfi DNA ligase and deletion mutants. The adenylation activity was determined by DNA ligase[32 P]NADþ product on 10% SDS–polyacrylamide gel electrophoresis as described in Section 2. (b) DNA binding of wild-type Tfi DNA ligase and deletion mutants. The DNA binding activity was determined by retardation band of DNA ligase–DNA complex on 10% non-denaturing gel electrophoresis as described in Section 2. (c) Ligation assay of wild-type Tfi DNA ligase and deletion mutants. The nickclosing activity was determined by ligating 32 P-labeled nicked substrate as described in Section 2. Lane 1, wild-type Tfi DNA ligase; lane 2, TfiM1 mutant; lane 3, Tfi-M2 mutant; lane 4, Tfi-M3 mutant; lane 5, Tfi-M4 mutant; lane 6, Tfi-M5 mutant; lane 7, Tfi-M6 mutant; lane 8, Tfi-M7 mutant.
domain of NADþ -dependent DNA ligases is important to the reactivity of the enzymes with NADþ , and several residues within the domain Ia comprise a binding site for the nicotinamide mononucleotide moiety of NADþ [16,19]. The mutant Tfi-M4 is minimal domain containing the adenylation residue, Lys116, and domain Ia (residues 1–73) of Tfi DNA ligase. This result suggests that the adenylation of Tfi DNA ligase can occur independently for other domains and DNA binding, and the adenylation of Tfi-M4 domain is the first step of DNA ligation. Our self-adenylation results are consistent with the previous reports that Nterminal fragments of Bacillus stearothermophilus [20,21], Staphylococcus aureus [22] and Aquifex pyro-
To characterize the DNA binding activities of wildtype Tfi DNA ligase and seven deletion mutants, each type of DNA ligase was incubated with 32 P-labeled nicked duplex substrate and applied to native polyacrylamide gel electrophoresis (Fig. 3(b)). The wild-type and four deletion mutants (Tfi-M1, Tfi-M5, Tfi-M6 and Tfi-M7) could form enzyme–DNA complex. This result indicates that the C-terminal region of Tfi DNA ligase is responsible for the DNA binding activity. It was proposed that two distinct putative DNA binding sites exist in Tfi DNA ligase [9]; one is related to the OB-fold domain (domain 2) and the other is to the HhH motif of domain 3. The DNA binding activity of Tfi-M6 mutant, together with the result in Tfi-M2 mutant with no DNA binding activity, suggests that the zinc finger and HhH motif domain (domain 3), particularly the HhH motif, is important for the DNA substrate binding; however, the higher activity of Tfi-M5 mutant, compared to that of Tfi-M6 mutant, in the DNA binding suggests that the OB-fold domain is needed for the maximum DNA binding activity of Tfi DNA ligase. The Tfi-M1 deleting only the BRCT domain showed not only DNA binding activity but also nick-closing activity (described in Section 3.4), suggesting that the unexpected DNA binding activity of Tfi-M7 mutant might not be related directly to the substrate binding occurred during DNA ligation. Our suggestions are largely consistent with the results of limited proteolysis studies on B. stearothermophilus [20] and S. aureus [22] NADþ -dependent DNA ligases. It was shown that the DNA binding activity of the Cterminal fragments, corresponding to domains 3 and 4 of Tfi DNA ligase, of B. stearothermophilus (residues 397–670) and S. aureus (residues 391–667) DNA ligases is comparable to the full-length protein, respectively, and the activity of these fragments is independent of the N-terminal fragments (residues 1–318 in B. stearothermophilus DNA ligase and residues 1–315 in S. aureus DNA ligase), which are responsible for self-adenylation [20,22]. Both N-terminal fragments, corresponding to domain 1 of Tfi DNA ligase, have no DNA binding activity. This suggests that domain 3 plays an important role in DNA binding and that domain 1 alone is not sufficient for DNA binding. Our results suggest that the inability of the N-terminal fragments of B. stearothermophilus and S. aureus DNA ligases to bind strongly to duplex DNA may be due to the loss of the OB-fold domain (domain 2) that occurred during limited proteolysis, because this domain provides one side of the catalytic DNA binding site [9].
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3.4. Nick-closing activity and genetic complementation test The nick-closing activities of wild-type Tfi DNA ligase and seven deletion mutants were assayed using both 32 P-labeled and non-hazardous labeled nicked duplex substrates. Among the seven deletion mutants, only Tfi-M1 mutant showed the ligation activity for nick-closing in the ligation assay using 32 P-labeled substrate (Fig. 3(c)); however, the ratio of the ligation product by Tfi-M1 mutant to that by wild-type Tfi DNA ligase was approximately 63% as determined by densitometric evaluation. The results were consistent with those on the nick-closing activity assay using non-radiolabeled substrate; only Tfi-M1 mutant catalyzed the nick-closing reaction, showing the lower activity compared to the wild-type Tfi DNA ligase (described in Section 3.5). For the genetic complementation test, recombinant plasmids pTFM1, pTFM2, pTFM3 and pTFM4, which express Tfi DNA ligases deleting 86, 219, 264 and 353 amino acid residues from the C-terminus, were transformed into E. coli GE1720 cells, respectively. Among the four C-terminal deletion mutants, transformant harbouring pTFM1, which express the Tfi-M1 mutant, was only able to grow at 42 °C (Fig. 4). These in vitro and in vivo results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the DNA ligase activity, and the remainder of among their domains are essential for the DNA ligase activity. The BRCT (BRCA1 carboxyl terminus) domain is a widely duplicated sequence module recently identified by homology shared between more than 40 proteins, many of which are central to DNA repair and cell cycle control processes [23,24]. The BRCT domain present in NADþ -dependent DNA ligase is a distinct version of its kind and is shared by the large subunits of eukaryotic replication factor C and PARP [23]. Evolutionarily, it must be the ancestor of eukaryotic BRCT domains. It
was suggested that BRCT domains are likely to perform critical functions in the cell cycle control of organisms from bacteria to humans [23]. To determine the role of BRCT domain, toward understanding its apparent role in DNA repair or associated cellular functions, two transformants of E. coli GE1720 harbouring pTFL (wild-type) and pTFM1 (Tfi-M1 mutant) were streaked to LB plate containing IPTG, irradiated by UV lamp under same conditions, and then incubated at 42 °C, respectively. We thought that the growth rate of the cells harbouring pTFM1 was slow than that of the cells harbouring pTFL. However, the cells harbouring pTFM1 have no significant difference on the growth rate and external appearance compared to the cells harbouring pTFL. Thus, the role of BRCT domain of Tfi DNA ligase in the cell remains to be proved. 3.5. Enzymatic properties of wild-type Tfi DNA ligase and Tfi-M1 mutant The properties of wild-type Tfi DNA ligase and TfiM1 mutant were determined quantitatively by nickclosing activity assay using non-radiolabeled substrate. The enzymatic properties of Tfi-M1 mutant were slightly different from those of wild-type Tfi DNA ligase. The effect of temperature on enzymatic activity was determined within a range of 40–85 °C in 50 mM Tris–HCl (pH 8.0). The temperature profile of Tfi-M1 mutant was very similar to wild-type Tfi DNA ligase, reaching a maximal activity at 70 °C, but catalytic activity was approximately 50% compared with that of wild-type Tfi DNA ligase (Fig. 5(a)). The nick-closing activity of the enzymes was tested at two different temperatures: 70 and 90 °C (Fig. 5(b)). The wild-type Tfi DNA ligase was comparatively stable at 70 °C. The activity of the enzyme decreased slowly at 90 °C. In the case of Tfi-M1 mutant, the activity of the enzyme dropped fast at 90 °C. In short, this result suggests that the BRCT domain of Tfi DNA ligase contributes the structural stabilization
Fig. 4. In vivo complementation of an E. coli GE1720 cells (ligts251 mutant) using wild-type Tfi DNA ligase and deletion mutants. The strains harbouring the indicated plasmids were streaked on LB plates containing 50 lg ml1 of ampicillin and 1 mM IPTG, and incubated at 30 °C (a) or 42 °C (b). pJR, control vector; pTFL, wild-type Tfi DNA ligase; pTFM1, Tfi-M1 mutant (residues 1–581); pTFM2, Tfi-M2 mutant (residues 1–448); pTFM3, Tfi-M3 mutant (residues 1–403); pTFM4, Tfi-M4 mutant (residues 1–314).
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Fig. 5. Properties of wild-type Tfi DNA ligase and Tfi-M1 mutant. The enzyme activity was assayed at the indicated temperature and pH in the presence of 5 mM MgCl2 . Relative activity was expressed as a percentage of the maximum activity of the wild-type Tfi DNA ligase obtained. (a) Effect of temperature on wild-type Tfi DNA ligase (j) and Tfi-M1 mutant (d) activity in 50 mM Tris–HCl (pH 8.0). (b) Thermostability of wild-type Tfi DNA ligase (j, ) and Tfi-M1 mutant (d, s) at 70 °C (j, d) and 90 °C (, s). (c) Effect of pH on wild-type Tfi DNA ligase (j, ) and Tfi-M1 mutant (d, s) activity in 50 mM MES (j, d) and 50 mM Tris–HCl (, s).
for thermal conditions and catalytic activity of the enzyme. The dependence of wild-type Tfi DNA ligase and TfiM1 mutant on the pH was determined between pH 5.5– 9.0. The optimum pH of Tfi-M1 mutant changed from 7.0 to 8.5, and the enzyme had a narrow pH activity spectrum than that of wild-type Tfi DNA ligase (Fig. 5(c)). In optimum condition, the enzyme activity of Tfi-M1 mutant was also approximately 50% compared with that of wild-type Tfi DNA ligase. The effects of metal ions at 5 mM concentration were examined on the ligase activity of wild-type Tfi DNA ligase and Tfi-M1 mutant. Both activities of wild-type Tfi DNA ligase and Tfi-M1 mutant were activated by Mg2þ , Ca2þ and Mn2þ , but not by Co2þ , Zn2þ , Cu2þ and Ni2þ as well as EDTA (Table 1). This dependence of the catalytic activity on Mg2þ , Ca2þ and Mn2þ has al-
Table 1 Effects of various substances on the activities of wild-type Tfi DNA ligase and Tfi-M1 mutant Substance
MgCl2 CaCl2 MnCl2 CoCl2 ZnCl2 CuCl2 NiCl2 EDTA
Relative activity (%) Wild-type
Tfi-M1 mutant
100 34 23 4 6 8 9 0
100 35 21 8 5 7 8 0
Each substance was tested at concentration of 5 mM in standard nick-closing activity assay buffer.
ready been observed in NADþ -dependent DNA ligases from the genus Thermus, including T. thermophilus HB8 and T. species AK16D [25]. It should also be noted that divalent metal ions were necessary for catalysis. Comparing the influences of the three divalent cations, we found that wild-type Tfi DNA ligase and Tfi-M1 mutant exhibited higher activity in the presence of Mg2þ (Table 1). In general, Mg2þ is the preferred metal ion for DNA ligases [4,25]. Tong et al. repoted that T. thermophilus HB8 and T. species AK16D DNA ligases could use Mn2þ , instead of Mg2þ , to support ligation activity in reaction more than 20 min, however these enzymes were more active with Mg2þ than with Mn2þ in the time course and divalent cation concentration dependence tests [25]. In conclusion, the functions of the four distinct domains of NADþ -dependent Tfi DNA ligase were revealed by the biochemical assays of deletion mutants. The adenylation domain (domain 1) was essential for the self-adenylation, and the zinc finger and HhH motif domain (domain 3) and the OB-fold domain (domain 2) were important for the DNA binding. The three domains except the BRCT domain (domain 4) were needed for the nick-closing. Studies on the multidomains of Tfi DNA ligase using the deletion mutants proved more precisely the function of each domain determined from the crystal structure.
Acknowledgements This work was supported by a Grant-in Aid from the Korea Science and Engineering Foundation (KOSEF
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R05-2001-000-00534-0). We wish to thank Dr. Sigrıdur H. Thorbjarnard ottir, Institute of Biology, University of Iceland, Iceland, for the gift of E. coli GE1720.
References [1] Lehman, I.R. (1974) DNA ligase: structure, mechanism, and function. Science 186, 790–797. [2] Tong, J., Barany, F. and Cau, W. (2000) Ligation reaction specificities of an NADþ -dependent DNA ligase from the hyperthermophile Aquifex aeolicus. Nucl. Acids Res. 28, 1447–1454. [3] Barany, F. (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc. Natl. Acad. Sci. USA 88, 189–193. [4] Takahashi, M., Yamaguchi, E. and Uchida, T. (1984) Thermophilic DNA ligase. J. Biol. Chem. 259, 10041–10047. [5] Takahashi, M. and Uchida, T. (1986) Thermophilic HB8 DNA ligase: effects of polyethylene glycols and polyamines on blunt-end ligation of DNA. J. Biochem. 100, 123–131. [6] Barany, F. and Gelfand, D.H. (1991) Cloning, over-expression and nucleotide sequence of a thermostable DNA ligase-encoding gene. Gene 109, 1–11. [7] J onsson, Z.O., Thorbjarnardottir, S.H., Eggertsson, G. and Palsdottir, A. (1994) Sequence of the DNA ligase-encoding gene from Thermus scotoductus and conserved motifs in DNA ligases. Gene 151, 177–180. [8] Kim, H.-K. and Kwon, S.-T. (1998) Cloning, nucleotide sequence, and expression of the ligase-encoding gene from Thermus filiformis. Mol. Cells 8, 438–443. [9] Lee, J.Y., Chang, C., Song, H.K., Moon, J., Yang, J.K., Kim, H.K., Kwon, S.-T. and Suh, S.W. (2000) Crystal structure of NADþ dependent DNA ligase: modular architecture and functional implications. EMBO J. 19, 1119–1129. [10] Dermody, J.J., Robinson, G.T. and Sternglanz, R. (1979) Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli. J. Bacteriol. 139, 701–704. [11] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [12] Kwon, S.-T., Kim, J.S., Park, J.H., Kim, H.K. and Lee, D.-S. (1997) Cloning and analysis of DNA polymerase-encoding gene from Thermus caldophilus GK24. Mol. Cells 7, 264–271.
[13] Weber, K., Pringle, J.R. and Osborn, M. (1971) Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Meth. Enzymol. 26, 3–27. [14] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. [15] Sriskanda, V. and Shuman, S. (1998) Chlorella virus DNA ligase: nick recognition and mutational analysis. Nucl. Acids Res. 26, 525–531. [16] Lim, J.-H., Choi, J., Kim, W., Ahn, B.-Y. and Han, Y.S. (2001) Mutational analyses of Aquifex pyrophilus DNA ligase define essential domains for self-ligation and DNA binding activity. Arch. Biochem. Biophys. 388, 253–260. [17] Thorbjarnard ottir, S.H., J onsson, Z.O., Andresson, O.S., Kristjansson, J.K., Eggertsson, G. and Palsdottir, A. (1995) Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases. Gene 161, 1–6. [18] Luo, J. and Barany, F. (1996) Identification of essential residues in Thermus thermophilus DNA ligase. Nucl. Acids Res. 24, 3079– 3085. [19] Sriskanda, V. and Shuman, S. (2002) Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NADþ . J. Biol. Chem. 277, 9695–9700. [20] Timson, D.J. and Wigley, D.B. (1999) Functional domains of an NADþ -dependent DNA ligase. J. Mol. Biol. 285, 73–83. [21] Singleton, M.R., Hakansson, K., Timson, D.J. and Wigley, D.B. (1999) Structure of the adenylation domain of an NADþ dependent DNA ligase. Structure 7, 35–42. [22] Kaczmarek, F.S., Zaniewski, R.P., Gootz, T.D., Danley, D.E., Mansour, M.N., Griffor, M., Kamath, A.V., Cronan, M., Mueller, J., Sun, D., Martin, P.K., Benton, B., McDowell, L., Biek, D. and Schmid, M.B. (2001) Cloning and functional characterization of an NADþ -dependent DNA ligase from Staphylococcus aureus. J. Bacteriol. 183, 3016–3024. [23] Bork, P., Hofmann, K., Bucher, P., Neuwald, A.F., Altschul, S.F. and Koonin, E.V. (1997) A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J. 11, 68–76. [24] Callebaut, I. and Mornon, J.P. (1997) From BRCA1 to RAP1: a widespread BRCT module closely associated with DNA repair. FEBS Lett. 400, 25–30. [25] Tong, J., Cao, W. and Barany, F. (1999) Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D. Nucl. Acids Res. 27, 788–794.
Mutational analyses of the thermostable NADþ-dependent DNA ligase from Thermus filiformis Hyo Jeong Jeon a, Hea-Jin Shin a, Jeong Jin Choi a, Hyang-Sook Hoe a, Hyun-Kyu Kim b, Se Won Suh c, Suk-Tae Kwon a,* a
Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea b Genetic Resource R&D Institute, Super Bio Co., Ltd., Suwon 440-746, Republic of Korea c Department of Chemistry, Seoul National University, Seoul 151-742, Republic of Korea Received 17 February 2004; received in revised form 19 May 2004; accepted 14 June 2004 First published online 22 June 2004
Abstract The crystal structure of NADþ -dependent DNA ligase from Thermus filiformis (Tfi) revealed that the protein comprised four structural domains. In order to investigate the biochemical activities of these domains, seven deletion mutants were constructed from the Tfi DNA ligase. The mutants Tfi-M1 (residues 1–581), Tfi-M2 (residues 1–448), Tfi-M3 (residues 1–403) and Tfi-M4 (residues 1– 314) showed the same adenylation activity as that of wild-type. This result indicates that only the adenylation domain (domain 1) is essential for the formation of enzyme–AMP complex. It was found that the zinc finger and helix-hairpin-helix (HhH) motif domain (domain 3) and the oligomer binding (OB)-fold domain (domain 2) are important for the formation of enzyme–DNA complex. The mutant Tfi-M1 alone showed the activities for in vitro nick-closing and in vivo complementation in Escherichia coli as those of wildtype. These results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the enzyme activity. The enzymatic properties of Tfi-M1 mutant (deleted the BRCT domain) were slightly different from those of wild-type and the nickclosing activity of Tfi-M1 mutant was approximately 50% compared with that of wild-type. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Thermus filiformis (Tfi) DNA ligase; Self-adenylation; DNA binding activity; Nick-closing activity
1. Introduction DNA ligases (EC 6.5.1.1 and EC 6.5.1.2) catalyze the sealing of 50 -phosphate and 30 -hydroxyl termini at single-strand breaks in double-stranded DNA, or at two fragments containing either complementary single strand or blunt ends, which are essential in DNA replication, recombination and repair [1]. Early studies on Escherichia coli and T4 DNA ligases have demonstrated the different cofactor requirements and elucidated the basic enzymatic mechanism of DNA ligation. ATP-
*
Corresponding author. Tel.: +82-31-290-7863; fax: +82-31-2907870. E-mail address: [email protected] (S.-T. Kwon).
dependent DNA ligases have been found in bacteriophages, archaea, eukaryotes, and recently in bacteria, whereas NADþ -dependent DNA ligases have exclusively been found in bacteria [2]. Apart from the difference in cofactor requirement, the reactions catalyzed by the two classes of DNA ligase are identical. In the first step, an AMP group derived from either cofactor is covalently attached to the conserved lysine residue within the Lys–Xaa–Asp–Gly (KXDG) motif. The AMP moiety is then transferred from the adenylated enzyme intermediate to the free 50 -phosphoryl group at a nicked site of duplex DNA. Finally, the AMP group is released from the adenylated DNA intermediate as the phosphodiester bond is formed [1]. Bacterial ligases from thermophilic organisms have become model systems for structural and mechanistic
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.06.018
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studies of DNA ligation due to their high thermostability. Recently, the thermostable DNA ligase is the key component of the ligase chain reaction (LCR) for amplifying and detecting a single-base genetic disease [3]. Studies on the properties of Thermus thermophilus HB8 (Tth) DNA ligase have been first reported among thermostable DNA ligases [4,5]. This enzyme is active at temperature above 70 °C. The genes encoding Tth DNA ligase [6], T. scotoductus DNA ligase [7] and T. filiformis (Tfi) DNA ligase [8] were cloned into E. coli, respectively, and their nucleotide sequences and deduced amino acid sequences were reported. Especially, Tfi DNA ligase gene contains an open reading frame encoding 667 amino acids with a molecular weight of 75,936 Da [8]. The crystal structure of Tfi DNA ligase, a 667-residue multidomain protein, has recently been solved [9]. Tfi DNA ligase was found to have a unique circular arrangement of its four distinct domains, domain 1 (residues 1–317, adenylation domain), domain 2 (residues 318–403, oligomer binding (OB)-fold domain), domain 3 (residues 404–581, zinc finger and helix-hairpin-helix (HhH) motif domain) and domain 4 (residues 582–667, BRCT domain), which leads to a hole large enough to hold a double-strand DNA. In this paper, we report the cloning and the purification of seven deletion mutants of Tfi DNA ligase, and the biochemical characteristics of the deletion mutants, to define the essential domain(s) for self-adenylation, DNA binding activity and nick-closing activity of the NADþ -dependent Tfi DNA ligase.
2. Materials and methods 2.1. Bacterial strains E. coli BL26Blue {ompT hsdSB (rB mB ) gal dcm lac [F0 proAB lacIq Z DM15:: Tn10 (TcR )]} was used as the host for plasmid preparations and gene expression. E. coli GE1720 (provided by Dr. Sigrıdur H. Thorbjarnard ottir, University of Iceland) was used as the host for in vivo complementation test of DNA ligase activity. This strain carries the ligts251 mutation which had previously been shown to cause loss of viability at 41 °C due to inability to perform ligation of Okazaki fragments [10]. 2.2. Cloning of the deletion mutants of Tfi DNA ligase Standard procedures for DNA ligation, transformation and other cloning-related techniques were as described by Sambrook et al. [11]. The oligonucleotides used as primers were synthesized to clone deletion mutants based on the nucleotide sequence of Tfi DNA ligase gene [8] and the crystal structure of Tfi DNA ligase [9]. The sense primers added a unique EcoRI site
(underlined), and the antisense primers added a unique SalI site (underlined). Recombinant plasmids pTFM1, pTFM2, pTFM3 and pTFM4, which express Tfi DNA ligases deleting 86, 219, 264 and 353 amino acid residues from the C-terminus (designate as Tfi-M1 (residues 1– 581), Tfi-M2 (residues 1–448), Tfi-M3 (residues 1–403) and Tfi-M4 (residues 1–314), respectively), were constructed by a method similar to that described previously [8]. PCR was done with pTFL (contains the Tfi DNA ligase gene)[8] as a template using a common sense primer of 50 -AGACTGAATTCATGACCCGGGAAGA-30 (N-terminal region) and four different antisense primers, that is, 50 -NNNNGTCGACTCACTTGGACTCCATGCTCAC-30 (C-terminal region of Tfi-M1), 50 -NNNNGTCGACTCAGCCCTCTATGTCCATG-30 (C-terminal region of Tfi-M2), 50 -NNNNGTCGACTCAGGGCCAGCGGATGGGCCG-30 (C-terminal region of Tfi-M3), and 50 -NNNNGTCGACTCAGGGGAACTTGTAGGCGAG-30 (Cterminal region of Tfi-M4). Recombinant plasmids pTFM5, pTFM6 and pTFM7, which express Tfi DNA ligases deleting 314, 403 and 581 amino acid residues from the N-terminus (designate as Tfi-M5 (residues 315– 667), Tfi-M6 (residues 404–667) and Tfi-M7 (residues 582–667), respectively), were also constructed. PCR was done with pTFL [8] as a template using a common antisense primer of 50 -CTGGGGTCGACTCAGGCCGGGACGG-30 (C-terminal region) and three different sense primers, that is, 50 -NNNNGAATTCATGGCCGAGGAGAAGGAGACC-30 (N-terminal region of Tfi-M5), 50 -NNNNGAATTCATGGAGGCCTGTCC CGAGTGC-30 (N-terminal region of Tfi-M6), and 50 -NNNNGAATTCATGGAGGAGGTCTCGGAC-30 (N-terminal region of Tfi-M7). Each of the PCR products was digested with EcoRI and SalI, and ligated into the expression vector pJR [12] that had been digested with the same enzymes, giving a fusion that used the tac promoter. E. coli BL26Blue was transformed with the ligates, respectively. Each of the all seven recombinant plasmids was identified by screening a transformants.
2.3. Purification of wild-type and mutants of Tfi DNA ligase expressed in E. coli Ten ml of an overnight culture of E. coli BL26Blue harbouring the recombinant plasmid grown in an Lbroth containing ampicillin were transferred to 1 l of the same medium and cultured at 37 °C, respectively. When the A600 of the culture was about 0.8, the culture was induced by the addition of isopropyl-b-D -thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM, and then incubated at 37 °C for another 6 h. The cells were collected by centrifugation, resuspended in 20 ml of sonication buffer (10 mM Tris–HCl, pH 7.6, and 2 mM MgCl2 ) containing 1 mM phenylmethylsulfonyl fluoride
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(PMSF), then disrupted by sonication. The disrupted cells were centrifuged at 10,000g, 4 °C for 20 min to remove E. coli cell debris. The sonicated extract was incubated at 80 °C for 30 min. The nucleic acids in the supernatant were precipitated by the addition of 1% streptomycin sulfate at room temperature with stirring for 30 min, then the precipitate was removed by centrifugation. The supernatant was dialyzed against buffer A (10 mM Tris–HCl, pH 7.6, 1 mM EDTA and 1 mM PMSF), then applied onto a DEAE–Sephacel column (1.5 11.3 cm) that had been equilibrated with buffer A. The adsorbed protein was eluted with a linear gradient of KCl (0–0.5 M) prepared in 120 ml of buffer A. The polypeptide composition of the column fractions was monitored by SDS–PAGE [13] on a 10% polyacrylamide gel and the DNA ligase fractions were pooled and dialyzed. Protein concentration was determined by the procedure of Lowry et al. [14] with bovine serum albumin (BSA) as a standard. 2.4. Adenylation assay The adenylation reactions were performed by incubating 100 pmol of Tfi DNA ligase and deletion mutants with 1 lCi [32 P]NADþ (800 Ci/mmol, Amersham Biosciences, UK) in a 10 ll reaction mixture (20 mM Tris– HCl, pH 8.0, 10 mM KCl, 5 mM MgCl2 and 2 mM DTT) at 70 °C for 10 min. The reaction was stopped by boiling in SDS loading buffer for 5 min and was analyzed by electrophoresis on a 10% SDS–polyacrylamide gel. The gel was dried, and the adenylated protein bands were analyzed by autoradiography. 2.5. Ligase substrate The substrate used in DNA binding and ligation assays was a 50 bp DNA duplex containing a centrally placed nick. The DNA duplex was formed by annealing two 25mer oligodeoxyribonucleotides to a complementary 50mer template strand as follows. The 3MP18 oligomer (50 -ATCCCCGGGTACCGAGCTCGAATTC-30 , 50 pmol) complementary to the 50 side of the template was radiolabeled by T4 polynucleotide kinase in the presence of 200 lCi [c-32 P]ATP (3000 Ci/mmol, Amersham Biosciences, UK) at 37 °C for 1 h, and purified by ethanol precipitation with ammonium acetate. The labeled oligomer was annealed to the template strand (mp18-Tem, 50 -GAATTCGAGCTCGGTACCCGGG GATCCTCTAGAGTCGACCTGCAGGCATG-30 ) in the presence of the 5MP18 oligomer (50 -CATGCCTGCAGGTCGACTCTAGAGG-30 ), com- plementary to the 30 side of the template, in annealing buffer (20 mM Tris–HCl, pH 8.0, and 10 mM KCl). The mixture was slowly cooled to room temperature after heating at 95 °C for 5 min. The molar ratio of the components,
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3MP18, 5MP18 and mp18-Tem in annealing mixture was 1:1.2:1.2, respectively. 2.6. DNA binding and ligation assays For DNA binding assay, reaction mixtures (10 ll) containing 20 mM Tris–HCl, pH 8.0, 10 mM KCl, 2 mM DTT, 0.2 pmol of 32 P-labeled nicked duplex substrate and 100 pmol of Tfi DNA ligase and deletion mutants were incubated at 50 °C for 30 min [15,16]. The reacted samples were electrophoresed through a native 10% polyacrylamide gel in TBE (90 mM Tris–borate and 2.5 mM EDTA) at 70 V for 2 h. Ligase–DNA complexes were visualized by autoradiography of the dried gel. For DNA ligation assay, reaction mixtures (10 ll) containing 20 mM Tris–HCl, pH 8.0, 10 mM KCl, 5 mM MgCl2 , 2 mM DTT, 0.5 mM NADþ , 0.2 pmol of 32 P-labeled nicked duplex substrate and 100 pmol of Tfi DNA ligase and deletion mutants were incubated at 60 °C for 1 h [15,16]. After incubation, reactions were halted by addition of EDTA and formamide. The reacted samples were heated at 95 °C for 5 min, and then electrophoresed through a 15% polyacrylamide gel containing 7 M urea in TBE. The ligation products were visualized by autoradiography of the dried gel. 2.7. Nick-closing activity assay The assay for nick-closing activity of DNA ligase was performed by the method of Thorbjarnard ottir et al. [17]. The sequences of oligodeoxyribonucleotides used in substrate preparation for nick-closing activity assay were the same as described above; however, the 3MP18 oligomer was labeled with biotin at 30 -end and phosphorylated at 50 -end, and the 5MP18 oligomer was labeled with fluorescein at 50 -end. Tfi DNA ligase and deletion mutants (0.2 pmol) were mixed with 5 pmol of the annealed substrate in 20 ll of standard assay buffer (20 mM Tris–HCl, pH 8.0, 10 mM KCl, 5 mM MgCl2 , 0.5 mM NADþ and 0.01% BSA) and overlayed with paraffin oil. The DNA ligase activity was assayed at 70 °C for 5 min. The reactions were terminated by the addition of 2 ll of 0.1 M EDTA and incubated on ice for 10 min. Triplicate aliquots of 5 ll from each reacted sample were then applied to wells in streptavidin coated 96-well microtiter plate (MaxiSorp; Nunc, Denmark) with 45 ll of washing buffer (100 mM Tris–HCl, pH 8.0, 150 mM NaCl and 0.05% Tween20). The plate was incubated at 37 °C for 1 h, washed once with sterilized water, twice with denaturation buffer (0.1 N NaOH and 0.05% Tween20), twice with sterilized water, once more with washing buffer, and incubated at 37 °C for 30 min with 50 ll of a 1:3000 diluted anti-fluorescein-alkaline phosphatase conjugate (Boehringer-Mannheim, Germany) in washing buffer. After washing six times with
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washing buffer, 100 ll of fresh substrate solution (5 mM p-nitrophenyl phosphate, 100 mM Tris–HCl, pH 9.0, 1 mM MgCl2 and 0.5 mM ZnCl2 ) were added and incubated at 37 °C for 10 min. The resulting yellow color was read by a microplate reader at 405 nm.
3. Results and discussion 3.1. Cloning and purification of the deletion mutants of Tfi DNA ligase A total of seven deletion mutants were constructed based on the X-ray crystal structure of Tfi DNA ligase [9] (Fig. 1). To define the function of the multidomains, four C-terminal deletion mutants, Tfi-M1 (residues 1– 581), Tfi-M2 (residues 1–448), Tfi-M3 (residues 1–403) and Tfi-M4 (residues 1–314), and three N-terminal deletion mutants, Tfi-M5 (residues 315–667), Tfi-M6 (residues 404–667) and Tfi-M7 (residues 582–667), were constructed. The wild-type Tfi DNA ligase and deletion mutants were expressed in E. coli BL26Blue as soluble proteins. The expression level was approximately 5–9% of the total E. coli protein for the wild-type Tfi DNA ligase and deletion mutants. The expressed cells were harvested and ultrasonicated. The proteins were then purified by heat treatment (to denature the E. coli proteins) and one step of DEAE–Sephacel column chromatography. The purified proteins were above 95% pure as judged by densitometric evaluation of proteins separated by SDS–PAGE (Fig. 2). 3.2. Analysis of the enzyme-AMP formation The adenylation of wild-type Tfi DNA ligase and seven deletion mutants were compared with one an-
Fig. 2. SDS–PAGE analysis of wild-type Tfi DNA ligase and deletion mutants. The purified proteins were analyzed on 10% SDS–polyacrylamide gel and stained with Coomassie brilliant blue R-250. Lane 1, wild-type Tfi DNA ligase; lane 2, Tfi-M1 mutant (residues 1–581); lane 3, Tfi-M2 mutant (residues 1–448); lane 4, Tfi-M3 mutant (residues 1–403); lane 5, Tfi-M4 mutant (residues 1–314); lane 6, Tfi-M5 mutant (residues 315–667); lane 7, Tfi-M6 mutant (residues 404–667); lane 8, Tfi-M7 mutant (residues 582–667); lane M, low-molecularweight markers (molecular masses are indicated at the right).
other by incubating each type of DNA ligase in the presence of [32 P]NADþ , followed by SDS–PAGE (Fig. 3(a)). It was shown that the wild-type and four Cterminal deletion mutants formed enzyme–AMP complexes. This result indicates that the N-terminal adenylation domain (domain 1) is essential for the formation of enzyme–AMP complex. The conserved lysine residue within the Lys–Xaa–Asp–Gly motif has been reported as the adenylation residue for the selfadenylation of DNA ligases [15,18]. We also proposed that the adenylation site of Tfi DNA ligase is the Lys116 of Lys–Val–Asp–Gly motif [8,9]. Recently, it has been reported that domain Ia within the adenylation
Fig. 1. Schematic representation of the deletion mutants of Tfi DNA ligase. Domains are in different sizes [9]: domain 1 (residues 1–317, adenylation domain), domain 2 (residues 318–403, OB-fold domain), domain 3 (residues 404–581, zinc finger and HhH motif domain) and domain 4 (residues 582–667, BRCT domain). Solid bars indicate the wild-type (Tfi-WT) and seven deletion mutants (Tfi-M1, M2, M3, M4, M5, M6 and M7) of Tfi DNA ligase.
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philus [16] NADþ -dependent DNA ligases composed solely of the adenylation domain retain full adenylation activity, unlike ATP-dependent DNA ligases. 3.3. Analysis of DNA binding
Fig. 3. Biochemical activities of wild-type Tfi DNA ligase and deletion mutants. (a) Adenylation of wild-type Tfi DNA ligase and deletion mutants. The adenylation activity was determined by DNA ligase[32 P]NADþ product on 10% SDS–polyacrylamide gel electrophoresis as described in Section 2. (b) DNA binding of wild-type Tfi DNA ligase and deletion mutants. The DNA binding activity was determined by retardation band of DNA ligase–DNA complex on 10% non-denaturing gel electrophoresis as described in Section 2. (c) Ligation assay of wild-type Tfi DNA ligase and deletion mutants. The nickclosing activity was determined by ligating 32 P-labeled nicked substrate as described in Section 2. Lane 1, wild-type Tfi DNA ligase; lane 2, TfiM1 mutant; lane 3, Tfi-M2 mutant; lane 4, Tfi-M3 mutant; lane 5, Tfi-M4 mutant; lane 6, Tfi-M5 mutant; lane 7, Tfi-M6 mutant; lane 8, Tfi-M7 mutant.
domain of NADþ -dependent DNA ligases is important to the reactivity of the enzymes with NADþ , and several residues within the domain Ia comprise a binding site for the nicotinamide mononucleotide moiety of NADþ [16,19]. The mutant Tfi-M4 is minimal domain containing the adenylation residue, Lys116, and domain Ia (residues 1–73) of Tfi DNA ligase. This result suggests that the adenylation of Tfi DNA ligase can occur independently for other domains and DNA binding, and the adenylation of Tfi-M4 domain is the first step of DNA ligation. Our self-adenylation results are consistent with the previous reports that Nterminal fragments of Bacillus stearothermophilus [20,21], Staphylococcus aureus [22] and Aquifex pyro-
To characterize the DNA binding activities of wildtype Tfi DNA ligase and seven deletion mutants, each type of DNA ligase was incubated with 32 P-labeled nicked duplex substrate and applied to native polyacrylamide gel electrophoresis (Fig. 3(b)). The wild-type and four deletion mutants (Tfi-M1, Tfi-M5, Tfi-M6 and Tfi-M7) could form enzyme–DNA complex. This result indicates that the C-terminal region of Tfi DNA ligase is responsible for the DNA binding activity. It was proposed that two distinct putative DNA binding sites exist in Tfi DNA ligase [9]; one is related to the OB-fold domain (domain 2) and the other is to the HhH motif of domain 3. The DNA binding activity of Tfi-M6 mutant, together with the result in Tfi-M2 mutant with no DNA binding activity, suggests that the zinc finger and HhH motif domain (domain 3), particularly the HhH motif, is important for the DNA substrate binding; however, the higher activity of Tfi-M5 mutant, compared to that of Tfi-M6 mutant, in the DNA binding suggests that the OB-fold domain is needed for the maximum DNA binding activity of Tfi DNA ligase. The Tfi-M1 deleting only the BRCT domain showed not only DNA binding activity but also nick-closing activity (described in Section 3.4), suggesting that the unexpected DNA binding activity of Tfi-M7 mutant might not be related directly to the substrate binding occurred during DNA ligation. Our suggestions are largely consistent with the results of limited proteolysis studies on B. stearothermophilus [20] and S. aureus [22] NADþ -dependent DNA ligases. It was shown that the DNA binding activity of the Cterminal fragments, corresponding to domains 3 and 4 of Tfi DNA ligase, of B. stearothermophilus (residues 397–670) and S. aureus (residues 391–667) DNA ligases is comparable to the full-length protein, respectively, and the activity of these fragments is independent of the N-terminal fragments (residues 1–318 in B. stearothermophilus DNA ligase and residues 1–315 in S. aureus DNA ligase), which are responsible for self-adenylation [20,22]. Both N-terminal fragments, corresponding to domain 1 of Tfi DNA ligase, have no DNA binding activity. This suggests that domain 3 plays an important role in DNA binding and that domain 1 alone is not sufficient for DNA binding. Our results suggest that the inability of the N-terminal fragments of B. stearothermophilus and S. aureus DNA ligases to bind strongly to duplex DNA may be due to the loss of the OB-fold domain (domain 2) that occurred during limited proteolysis, because this domain provides one side of the catalytic DNA binding site [9].
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3.4. Nick-closing activity and genetic complementation test The nick-closing activities of wild-type Tfi DNA ligase and seven deletion mutants were assayed using both 32 P-labeled and non-hazardous labeled nicked duplex substrates. Among the seven deletion mutants, only Tfi-M1 mutant showed the ligation activity for nick-closing in the ligation assay using 32 P-labeled substrate (Fig. 3(c)); however, the ratio of the ligation product by Tfi-M1 mutant to that by wild-type Tfi DNA ligase was approximately 63% as determined by densitometric evaluation. The results were consistent with those on the nick-closing activity assay using non-radiolabeled substrate; only Tfi-M1 mutant catalyzed the nick-closing reaction, showing the lower activity compared to the wild-type Tfi DNA ligase (described in Section 3.5). For the genetic complementation test, recombinant plasmids pTFM1, pTFM2, pTFM3 and pTFM4, which express Tfi DNA ligases deleting 86, 219, 264 and 353 amino acid residues from the C-terminus, were transformed into E. coli GE1720 cells, respectively. Among the four C-terminal deletion mutants, transformant harbouring pTFM1, which express the Tfi-M1 mutant, was only able to grow at 42 °C (Fig. 4). These in vitro and in vivo results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the DNA ligase activity, and the remainder of among their domains are essential for the DNA ligase activity. The BRCT (BRCA1 carboxyl terminus) domain is a widely duplicated sequence module recently identified by homology shared between more than 40 proteins, many of which are central to DNA repair and cell cycle control processes [23,24]. The BRCT domain present in NADþ -dependent DNA ligase is a distinct version of its kind and is shared by the large subunits of eukaryotic replication factor C and PARP [23]. Evolutionarily, it must be the ancestor of eukaryotic BRCT domains. It
was suggested that BRCT domains are likely to perform critical functions in the cell cycle control of organisms from bacteria to humans [23]. To determine the role of BRCT domain, toward understanding its apparent role in DNA repair or associated cellular functions, two transformants of E. coli GE1720 harbouring pTFL (wild-type) and pTFM1 (Tfi-M1 mutant) were streaked to LB plate containing IPTG, irradiated by UV lamp under same conditions, and then incubated at 42 °C, respectively. We thought that the growth rate of the cells harbouring pTFM1 was slow than that of the cells harbouring pTFL. However, the cells harbouring pTFM1 have no significant difference on the growth rate and external appearance compared to the cells harbouring pTFL. Thus, the role of BRCT domain of Tfi DNA ligase in the cell remains to be proved. 3.5. Enzymatic properties of wild-type Tfi DNA ligase and Tfi-M1 mutant The properties of wild-type Tfi DNA ligase and TfiM1 mutant were determined quantitatively by nickclosing activity assay using non-radiolabeled substrate. The enzymatic properties of Tfi-M1 mutant were slightly different from those of wild-type Tfi DNA ligase. The effect of temperature on enzymatic activity was determined within a range of 40–85 °C in 50 mM Tris–HCl (pH 8.0). The temperature profile of Tfi-M1 mutant was very similar to wild-type Tfi DNA ligase, reaching a maximal activity at 70 °C, but catalytic activity was approximately 50% compared with that of wild-type Tfi DNA ligase (Fig. 5(a)). The nick-closing activity of the enzymes was tested at two different temperatures: 70 and 90 °C (Fig. 5(b)). The wild-type Tfi DNA ligase was comparatively stable at 70 °C. The activity of the enzyme decreased slowly at 90 °C. In the case of Tfi-M1 mutant, the activity of the enzyme dropped fast at 90 °C. In short, this result suggests that the BRCT domain of Tfi DNA ligase contributes the structural stabilization
Fig. 4. In vivo complementation of an E. coli GE1720 cells (ligts251 mutant) using wild-type Tfi DNA ligase and deletion mutants. The strains harbouring the indicated plasmids were streaked on LB plates containing 50 lg ml1 of ampicillin and 1 mM IPTG, and incubated at 30 °C (a) or 42 °C (b). pJR, control vector; pTFL, wild-type Tfi DNA ligase; pTFM1, Tfi-M1 mutant (residues 1–581); pTFM2, Tfi-M2 mutant (residues 1–448); pTFM3, Tfi-M3 mutant (residues 1–403); pTFM4, Tfi-M4 mutant (residues 1–314).
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Fig. 5. Properties of wild-type Tfi DNA ligase and Tfi-M1 mutant. The enzyme activity was assayed at the indicated temperature and pH in the presence of 5 mM MgCl2 . Relative activity was expressed as a percentage of the maximum activity of the wild-type Tfi DNA ligase obtained. (a) Effect of temperature on wild-type Tfi DNA ligase (j) and Tfi-M1 mutant (d) activity in 50 mM Tris–HCl (pH 8.0). (b) Thermostability of wild-type Tfi DNA ligase (j, ) and Tfi-M1 mutant (d, s) at 70 °C (j, d) and 90 °C (, s). (c) Effect of pH on wild-type Tfi DNA ligase (j, ) and Tfi-M1 mutant (d, s) activity in 50 mM MES (j, d) and 50 mM Tris–HCl (, s).
for thermal conditions and catalytic activity of the enzyme. The dependence of wild-type Tfi DNA ligase and TfiM1 mutant on the pH was determined between pH 5.5– 9.0. The optimum pH of Tfi-M1 mutant changed from 7.0 to 8.5, and the enzyme had a narrow pH activity spectrum than that of wild-type Tfi DNA ligase (Fig. 5(c)). In optimum condition, the enzyme activity of Tfi-M1 mutant was also approximately 50% compared with that of wild-type Tfi DNA ligase. The effects of metal ions at 5 mM concentration were examined on the ligase activity of wild-type Tfi DNA ligase and Tfi-M1 mutant. Both activities of wild-type Tfi DNA ligase and Tfi-M1 mutant were activated by Mg2þ , Ca2þ and Mn2þ , but not by Co2þ , Zn2þ , Cu2þ and Ni2þ as well as EDTA (Table 1). This dependence of the catalytic activity on Mg2þ , Ca2þ and Mn2þ has al-
Table 1 Effects of various substances on the activities of wild-type Tfi DNA ligase and Tfi-M1 mutant Substance
MgCl2 CaCl2 MnCl2 CoCl2 ZnCl2 CuCl2 NiCl2 EDTA
Relative activity (%) Wild-type
Tfi-M1 mutant
100 34 23 4 6 8 9 0
100 35 21 8 5 7 8 0
Each substance was tested at concentration of 5 mM in standard nick-closing activity assay buffer.
ready been observed in NADþ -dependent DNA ligases from the genus Thermus, including T. thermophilus HB8 and T. species AK16D [25]. It should also be noted that divalent metal ions were necessary for catalysis. Comparing the influences of the three divalent cations, we found that wild-type Tfi DNA ligase and Tfi-M1 mutant exhibited higher activity in the presence of Mg2þ (Table 1). In general, Mg2þ is the preferred metal ion for DNA ligases [4,25]. Tong et al. repoted that T. thermophilus HB8 and T. species AK16D DNA ligases could use Mn2þ , instead of Mg2þ , to support ligation activity in reaction more than 20 min, however these enzymes were more active with Mg2þ than with Mn2þ in the time course and divalent cation concentration dependence tests [25]. In conclusion, the functions of the four distinct domains of NADþ -dependent Tfi DNA ligase were revealed by the biochemical assays of deletion mutants. The adenylation domain (domain 1) was essential for the self-adenylation, and the zinc finger and HhH motif domain (domain 3) and the OB-fold domain (domain 2) were important for the DNA binding. The three domains except the BRCT domain (domain 4) were needed for the nick-closing. Studies on the multidomains of Tfi DNA ligase using the deletion mutants proved more precisely the function of each domain determined from the crystal structure.
Acknowledgements This work was supported by a Grant-in Aid from the Korea Science and Engineering Foundation (KOSEF
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R05-2001-000-00534-0). We wish to thank Dr. Sigrıdur H. Thorbjarnard ottir, Institute of Biology, University of Iceland, Iceland, for the gift of E. coli GE1720.
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