Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (220K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Asano, S.
Right arrow Articles by Horiuchi, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Asano, S.
Right arrow Articles by Horiuchi, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research Pages 1882-1889  

Filamentous phage replication initiator protein gpII forms a covalent complex with the 5[prime] end of the nick it introduced
Introduction
Materials And Methods
   Materials
   Detection of covalent complex between gpII and nicked DNA
   Determination of the amino acid residue that is bound to nicked DNA
   Amino acid sequencing of the tryptic peptide covalently bound to DNA
   Construction of tyrosine-replacement mutants of gpII
   In vivo complementation and competition assays
Results
   Detection of covalent complex made of gpII and nicked DNA as an intermediate for nicking reaction
   Covalent bond between tyrosine 197 of gpII and the 5[prime] end of the nick
   Characterization of Y197F mutant gpII
Discussion
Acknowledgements
References

Filamentous phage replication initiator protein gpII forms a covalent complex with the 5[prime] end of the nick it introduced

Filamentous phage replication initiator protein gpII forms a covalent complex with the 5[prime] end of the nick it introduced

Satoshi Asano1,+, Atsushi Higashitani1,2,* and Kensuke Horiuchi1,§

1Division of Microbial Genetics, National Institute of Genetics, Mishima 411-8540, Japan and 2Institute of Genetic Ecology, Tohoku University, Sendai 980-8577, Japan

Received December 31, 1998; Revised and Accepted March 3, 1999

ABSTRACT

Rolling circle type DNA replication is initiated by introduction of a nick in the leading strand of the origin by the initiator protein, which in most cases binds covalently to the 5[prime] end of the nick. In filamentous phage, however, such a covalent complex has not been detected. Using a suitable substrate and short reaction time, we show that filamentous phage initiator gpII forms a covalent complex with nicked DNA, which rapidly dissociates unless gpII is inactivated. A peptide-DNA complex was isolated from trypsin digest of the complex by ion-exchange column chromatography and gel filtration, and its peptide sequence was determined. The result indicated that gpII was linked to DNA by the tyrosine residue at position 197 from the N-terminus. The mutant protein in which this tyrosine was replaced by phenylalanine did not show any detectable activity to complement gene II amber mutant phage in vivo. In vitro, the mutant protein recognized the origin and bent DNA as well as the wild-type does, but failed to introduce a nick and to relax the superhelicity of cognate DNA.

INTRODUCTION

The replication initiator protein gpII (gene II protein) of filamentous phages (f1, M13, fd, etc.) is a multi-functional protein that plays several key roles in replication of the plus (viral) strand in a rolling circle fashion. It introduces a nick at a specific site on the plus-strand of negatively supercoiled replicative form DNA (form I) (1). The 3[prime]-hydroxyl end of the nick serves as the primer for replication. The nicking reaction is preceded by an ordered series of DNA conformational changes (2) which are induced by specific binding (3,4) of gpII to the replication origin. The conformational changes include successive bending of the origin DNA and duplex melting around the nicking site. The melting is dependent on negative superhelicity of DNA (2). Furthermore, gpII functions at steps beyond nicking; it prompts rep helicase-mediated unwinding of origin DNA for replication (5), and it cleaves and circularizes the displaced single strand upon replication termination (6). gpII also has a sequence-specific topoisomerase activity. In vitro, gpII converts ~40% of form I substrate molecules to a relaxed form (form IV) as a result of nicking-closing reaction, while the other ~60% of form I are nicked to yield open circles (form II). These reactions do not require any external energy source such as ATP (1,7,8).

In many other rolling circle type DNA replication systems, including isometric single-stranded DNA phage such as [phis]X174 (9,10) and small plasmids such as pT181 from Gram-positive bacteria (11,12), and in transfer replication of plasmids such as F or R100 (13), the initiator proteins, which introduce a specific nick at the origin, form a stable, covalent complex with the 5[prime] end of the nick they produced. In all the cases studied, it is one or two specific tyrosine residues of the initiator protein that form the covalent bond with the terminal 5[prime] phosphate at the nick. However, such covalent complex formation has not been detected in the case of gpII of filamentous phages (5).

In this paper, we show that a fraction of DNA molecules that were nicked by gpII is indeed covalently linked to a tyrosine residue at position 197 of gpII, indicating that this residue is the active center for the nicking reaction. Contrary to the other cases of rolling circle type DNA replication systems so far reported, the covalent bond formed by gpII dissociates rapidly unless gpII is inactivated.

MATERIALS AND METHODS

Materials

Bacterial strains, phages and plasmids used are listed in Table 1. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase and terminal deoxynucleotidyl transferase were obtained from TaKaRa Co., Ltd. 32P-labeled nucleotide triphosphates were from Amersham. Oligonucleotides were purchased from Bex Co., Ltd. Wild-type, G73A and Y197F gpIIs were prepared from Escherichia coli BL21(DE3) bearing pYO125 (Table 1), DH5 bearing both pDG117IIAH11 and placIqAH3 (Table 1) (8), and BL21(DE3) bearing pYO148, respectively (Table 1). These proteins were purified to near homogeneity as described previously (4,14). Origin-specific DNA binding activity and nicking activity of the purified gpIIs were measured in vitro by the methods previously described (2,14).

Table 1. E.coli strains, phages and plasmids

Detection of covalent complex between gpII and nicked DNA

Synthetic oligonucleotide substrates for nicking reaction were terminally labeled at 3[prime]- or 5[prime]-end by either terminal deoxynucleo-tidyl transferase and [[alpha]-32P]ddATP (dideoxyATP) or T4 poly-nucleotide kinase and [[gamma]-32P]ATP, respectively. After nicking by gpII, the reaction was stopped by adding an SDS-containing loading buffer and boiling for 5 min. The sample was electrophoresed on a 10% polyacrylamide gel containing 0.3% SDS. Autoradiography and quantification of radioactivity were carried out using a Fuji bas2000 image analyzer.

Determination of the amino acid residue that is bound to nicked DNA

An oligonucleotide internally labeled with 32P (15) was prepared as follows. A synthetic oligonucleotide (32mer) 5[prime]-AAT AGT GGA CTC TTG TTC CAA ACT GGA ACA AC, whose sequence is identical to nucleotides +1 to +32 (relative to the nicking site) of the f1 plus strand origin, was labeled at the 5[prime] end by T4 polynucleotide kinase and [[gamma]-32P]ATP. This oligonucleotide was mixed with two other oligonucleotides, a 10mer (5[prime]-CAC GTT CTT T) whose sequence is identical to nucleotides -10 to -1 of the plus strand and a 20mer (5[prime]-GTC CAC TAT TAA AGA ACG TG) whose sequence is complementary to nucleotides +10 to -10. The mixture was subjected to annealing conditions and ligated by T4 DNA ligase. The 42mer (-10 to +32 of the plus strand) internally labeled with 32P thus produced was purified from an 8% polyacrylamide-8 M urea gel, and was annealed to a 25mer (5[prime]-GTT CCA GTT TGG AAC AAG AGT CCA C) whose sequence is complementary to nucleotides +29 to +5 of the f1 DNA. The partially double-stranded oligonucleotide thus obtained was used as substrate for the nicking reaction by G73A gpII.

After the nicking reaction was carried out at 37°C for 30 s, the mixture was hydrolyzed with 6 N HCl at 110°C for 30 min. The hydrolyzed sample was lyophilized, dissolved in H2O, and subjected to paper electrophoresis at 1500V for 90 min in 5% acetic acid adjusted to pH 3.5 with pyridine. The paper was dried, phospho-amino acid markers were visualized by ninhydrin, and radioactive products were detected by autoradiography using a Fuji bas2000 image analyzer.

Amino acid sequencing of the tryptic peptide covalently bound to DNA

In order to maximize the yield of the gpII-DNA covalent complex, reaction conditions were carefully studied, which led us to adopt the following protocol. The partially double-stranded oligonucleotide substrate (43 pmol), which was labeled at the 3[prime] end of the plus strand with 32P, was pre-incubated with G73A gpII (560 pmol) in 20 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 5 mM DTT and 80 mM KCl at 37°C for 15 min. The mixture was then chilled, and after sitting on ice for 5 min, the nicking reaction was started by adding MgCl2 to a final concentration of 6 mM. After 30 s at 0°C, the reaction was stopped by adding SDS (a final concentration of 0.05%) and boiling for 5 min. Fifty such reactions were carried out in separate tubes and pooled for further analysis. After SDS was removed using Amupure DT column (Amersham), the mixture was digested with trypsin (a final concentration of 20 µg/ml) at 37°C overnight. The digest was loaded onto a MonoQ HR 5/5 column (Pharmacia) pre-equilibrated with buffer A [50 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 10 mM NaCl], and eluted with a linear gradient of NaCl (0.01-1 M in buffer A). Fractions containing radioactivity, which were eluted at ~650 mM NaCl, were pooled. The pooled fractions were desalted using a Fast Desalting column HR 10/10 (Pharmacia), and after concentration through the second MonoQ HR 5/5 column, further fractionated on a Superdex 75 HR 10/30 column (Pharmacia) in buffer A. Radioactive peak fractions were pooled, concentrated again through a MonoQ HR5/5 column, and used for peptide sequence analysis which was carried out by Apro Bio science Co., Ltd.

Construction of tyrosine-replacement mutants of gpII

A series of gene II mutants in which a tyrosine residue was replaced by phenylalanine (Table 1) were constructed by the PCR mutagenesis method as described by Ito et al. (16), using synthetic primers which carried mutant sequences. The nucleotide sequence of each mutant thus obtained was confirmed by Sanger’s chain-termination method using Sequenase (Amersham).

In vivo complementation and competition assays

K38 cells which carried pYO84 or its derivatives containing a tyrosine-replacement mutation in gene II were grown at 37°C overnight in LB medium containing 50 µg/ml kanamycin. 100 µl of the overnight culture were infected with ~500 p.f.u. of R86 (gene II amber mutant) or wild-type f1 at room temperature for 5 min, and plated on TY plates containing 0.1 mM IPTG. After incubation for 8 h, the number and morphology of phage plaques were scored.

RESULTS

Detection of covalent complex made of gpII and nicked DNA as an intermediate for nicking reaction

Using synthetic oligonucleotides as substrate, we have previously shown that the nicking reaction by gpII takes place efficiently, even in the absence of negative superhelicity, if the nicking region of the plus strand is single-stranded and the binding region for gpII is double-stranded (partially double-stranded substrate) (2). We therefore used partially double-stranded substrate to study whether any fraction of the DNA ends produced upon cleavage by gpII is covalently attached to gpII to form an intermediate complex. In the experiment shown in Figure 1, the substrate used was 5[prime] terminally-labeled 39mer or 3[prime] terminally-labeled (39+1)mer (including a residue added for 3[prime] end-labeling) whose sequence was identical to that of nucleotides -9 to +30 (relative to the nicking site) of the f1 plus strand. This oligonucleotide was hybridized to an unlabeled 25mer whose sequence was complementary to nucleotides +29 to +5 of the f1 DNA (Fig. 1A). The partially double-stranded substrate thus obtained was completely cleaved by gpII under the experimental conditions in <15 s to yield a 5[prime]-proximal 9mer (Fig. 1B, lane 2) and a 3[prime]-proximal 30mer [or (30+1)mer with a residue added for 3[prime]-end labeling] (Fig. 1B, lane 6). The results indicated that at least the majority of the nicked DNA was not covalently bound to gpII, since the resultant covalent complex is expected to move much slower on the gel than these DNA fragments.

   A

   B

   C

Figure 1. Cleavage of synthetic oligonucleotides by gpII. (A) Partially double-stranded synthetic oligonucleotide used as substrate. The gpII nicking site is indicated by an arrowhead. Horizontal arrows indicate repeated sequences [beta], [gamma] and [delta] for gpII binding (4). Nucleotides that are melted when in form I DNA by binding of gpII are indicated by outlined letters. (B) Analysis of nicking reaction products. 0.2 pmol of substrate was incubated with 2 pmol of gpII in 100 µl of a reaction buffer (20 mM Tris-Cl, pH 8.0, 5 mM MgCl2, 5 mM DTT, 80 mM KCl) at 37°C. At various times as indicated, a 10 µl aliquot was withdrawn, and analyzed on an 18% polyacrylamide-8 M urea gel. In lanes 1-4 and lanes 5-8, the substrate used was labeled with 32P at the 5[prime] and 3[prime] ends, respectively. Size of DNA fragments was determined by use of synthetic oligonucleotides as previously described (2). (C) Detection of covalent complex formation. The reaction conditions were the same as in (B) except that the samples were analyzed on a 10% polyacrylamide-0.3% SDS gel. Exposure time for autoradiography was 10 times longer than in (B). Lanes 1-6 and lanes 7-12 show the results obtained with 5[prime]- and 3[prime]-labeled substrate, respectively. The positions of the two bands observed on lanes 8-12 approximately correspond to positions for proteins of 60 and 50 kDa as determined by use of molecular size markers (not shown).

However, when the same amount of the reaction mixture was analyzed by SDS-containing 10% polyacrylamide gel electrophoresis, two 32P-labeled bands corresponding to positions for proteins of ~50 and 60 kDa were detected after about 10 times longer exposure (Fig. 1C). These bands were observed only when 3[prime] end-labeled substrate (lanes 7-12), and not 5[prime]-labeled substrate (lanes 1-6), was used. This observation suggested that gpII is covalently bound to the 5[prime] end of the nick in a small fraction of nicked DNA molecules. The complex was formed only under the conditions that allowed nicking of DNA by gpII (data not shown).

When unlabeled 39mer of the plus strand was annealed to 32P-labeled 25mer of the complementary strand and used as substrate, only the slower (position of ~60 kDa) of the two bands of the covalent complex was observed (data not shown). Thus, the faster moving band (~50 kDa) represents the complex made of gpII and the 3[prime] proximal part of the nicked plus strand, while the slower band is a result of re-annealing of the complementary strand to the plus strand in the complex, which probably occurred during entry into the gel.

The amount of the covalent complex produced corresponded to only ~3% of the total nicked molecules at the reaction time of 15 s (Fig. 1C, lane 8), and decreased upon further incubation (Fig. 1C, lanes 9-12). The decrease stopped upon addition of SDS (data not shown). We conclude that gpII covalently binds to the 5[prime] end of the nick it produced and rapidly dissociates this covalent bond.

Covalent bond between tyrosine 197 of gpII and the 5[prime] end of the nick

The plus-strand replication origin of filamentous phage consists of two domains: a core origin that is absolutely required for DNA replication and an adjacent replication enhancer that increases in vivo replication 30-100-fold (7,17). The latter contains three binding sites for the E.coli integration host factor (IHF) (18). Phage mutants that can replicate in the absence of either replication enhancer or IHF have been isolated, and they turned out to be mutants of gene II (19-21). In the course of screening of gene II mutants in the in vitro nicking reaction, we found that gpII from one of the replication enhancer-independent mutants, G73A (glycine at position 73 of gpII replaced by alanine; 8), yielded ~5-20-fold larger amounts of covalent complex compared to the wild-type gpII (data not shown). This mutant gpII cleaved the origin exactly at the same position as the wild-type gpII. We therefore used the G73A gpII to identify the amino acid residue that covalently binds the nicked end.

We prepared a partially double-stranded oligonucleotide substrate, in which the phosphorus atom at the nicking site was specifically labeled with 32P (Materials and Methods). After short incubation with G73A gpII, the reaction mixture was hydrolyzed in 6N HCl and was analyzed by paper electrophoresis (Materials and Methods). The result shown in Figure 2 indicates that the position of the radioactive product from the hydrolysate coincided with the position of o-phospho-tyrosine, not with the positions of o-phospho-serine and o-phospho-threonine. Since those are the only phosphorylated amino acids commonly found in proteins and stable in acid treatment, the result strongly suggests that a tyrosine residue(s) of gpII covalently bound to the 5[prime] end of the nicked DNA.

There are 15 tyrosine residues in gpII. To determine which tyrosyl residue(s) forms the covalent bond with nicked DNA, the partially double-stranded substrate which was labeled at the 3[prime] end of the plus strand was incubated with G73A gpII as described in Materials and Methods, and was completely digested with trypsin. The peptide-DNA covalent complex was isolated by a series of column chromatography by chasing radioactivity (Materials and Methods), and analyzed by Edman degradation using an automatic peptide sequencer. The results of the PTH-amino acid analyses of eight cycles are shown in Figure 3A. Cycles 1-4 clearly showed Thr, Leu, Val and Ala, respectively. At cycle 5, no usual PTH-amino acid was detected, and a unique peak (marked with an arrowhead in Fig. 3A) was observed. Cycles 6 and 7 clearly showed Leu and Lys, respectively. No PTH-amino acid was detected after cycle 7 (10 cycles were performed). Comparison of the amino acid sequence thus determined (Fig. 3B) with the tyrosine-containing tryptic peptides of gpII predicted from DNA sequence clearly pointed out a single and perfect match: Thr, Leu, Val, Ala, Tyr, Leu, Lys with the Tyr residue at position 197 (Fig. 3B). We conclude that the amino acid residue of gpII, which covalently binds to the 5[prime] end of the nick, is tyrosine at position 197 from the N-terminus.


Figure 2. Analysis of the gpII-DNA covalent complex after acid hydrolysis. Partially double-stranded oligonucleotide substrate (Fig. 1B) in which the phosphorus at the nicking site was specifically labeled with 32P was incubated with gpII, hydrolyzed in 6N HCl, and subjected to paper electrophoresis and autoradiographed (lane 2). Lane 1, a control without gpII. Lanes 3-5, markers visualized with ninhydrin: o-phospho-tyrosine (lane 3), o-phospho-serine (lane 4) and o-phospho-threonine (lane 5).


Characterization of Y197F mutant gpII

We constructed 15 gene II mutants in each of which one of the 15 tyrosine residues of gpII was replaced by phenylalanine (Materials and Methods). Activity of the mutated gpII to rescue a gene II amber mutation (UGA nonsense mutation) was measured in vivo (Table 2). Wild-type cells (K38) harboring a plasmid carrying gene II with a tyrosine replacement mutation under a tac promoter were tested for their ability to form plaques of a gene II amber mutant phage (R86) in the presence of IPTG. Eight mutants, Y71F, Y79F, Y221F, Y252F, Y303F, Y337F, Y345F and Y360F, were found to normally complement the growth of R86 at both 37 and 42°C. Three mutants, Y272F, Y333F and Y402F, showed temperature-sensitive phenotype: they complemented R86 at 37 but not at 42°C. Three mutants, Y49F, Y86F and Y129F, complemented R86 to some extent, but only more weakly than wild-type gpII, as judged by the number and/or the size of plaques. Only one mutant, Y197F, was totally incapable of complementing the amber mutation. These results are summarized in Table 2.

Table 2. In vivo complementation of gene II amber phage by gene II mutants on plasmid
a(-) indicates the absence of IPTG.
bNumber of plaques relative to that obtained on the strain carrying wild-type gene II are listed.

   A

   B

Figure 3. Peptide sequencing analysis of the covalent complex. Partially double-stranded oligonucleotide substrate labeled with 32P at the 3[prime] end of the plus strand was incubated with G73A gpII, and the mixture was digested with trypsin. The 32P-labeled peptide was purified through a series of column chromatography, and subjected to amino acid sequencing. For details see Materials and Methods. (A) Profiles of PTH- analysis for eight cycles. (B) Summary of the result of peptide sequencing and the gpII tryptic peptide identified as that covalently bound to DNA by the result of peptide sequencing. X indicates a modified residue. Trypsin cleavage sites are marked by arrowheads.

Overproduction of defective mutant gpII that can interact with the replication origin may interfere with the growth of the wild-type phage by competing with phage-encoded gpII. This was tested for the seven tyrosine-replacement mutants that were either temperature-sensitive, poor, or inactive in the complementation test of R86 phage described above. K38 cells harboring either of these mutant gene II on a plasmid were infected with wild-type f1 phage and plated. Among the seven mutants tested, only Y197F led to formation of turbid plaques (data not shown), suggesting that the defective Y197F gpII may interact with the origin.

This observation prompted us to purify Y197F gpII and to test in vitro for its specific binding to the origin and for its nicking activity. Purified Y197F gpII bound to the origin and formed non-covalent complexes, complex I and complex II (4), in the same manner as wild-type gpII (Fig. 4A). The concentrations of Y197F gpII required to form each complex were approximately the same as those of wild-type gpII. Gel mobility of each complex formed by Y197F gpII was same as that formed by wild-type gpII (Fig. 4A). This indicates that both gpIIs bent DNA to a similar extent, since it has been demonstrated by analysis of circularly permuted DNA fragments that the mobility shift of this fragment by binding of wild-type gpII is largely due to DNA bending (2). However, Y197F gpII could not introduce a nick into the origin, regardless of whether the substrate was negatively supercoiled plasmid DNA carrying f1 origin (pYO5) or 32P-labeled synthetic oligonucleotide (Fig. 4B and C).

Altogether, these results indicate that the tyrosine residue 197 of gpII plays the role of the active center for the nicking reaction.

DISCUSSION

Formation of a covalent complex between initiator protein and 5[prime] end of the nick introduced by the initiator has been well known for many rolling circle type DNA replication systems, including lytic phage [phis]X174, small plasmids such as pT181, and transfer replication of conjugative plasmids such as F or R100. In these cases the covalent complex is extremely stable so that essentially all the DNA molecules nicked in vitro by the initiator protein are found in the form of covalent complex.

Such a covalent complex has not been detected in spite of extensive efforts in the cases of filamentous phages (1) and a small number of plasmids including pMV158 (22). Furthermore, in the transposition reaction of phage Mu by the MuA protein, a novel mechanism of direct transesterification without involvement of covalent intermediate has been demonstrated by an elegant experiment on the chirality of phosphate by Mizuuchi and Adzuma (23). The same mechanism could have been assumed to be operating in the filamentous phage gpII reaction.

The data presented in this paper, however, show that gpII forms a covalent complex with the 5[prime] end of the nick it introduced. Contrary to the cases of rolling circle type DNA replication previously reported, the covalent complex in filamentous phage dissociated rapidly unless gpII was inactivated; i.e., gpII possesses activities both to form the covalent bond and to break it. This could explain why the covalent complex of gpII had not been detected for a long time. Use of short reaction time and of sensitive substrate in which the nicking region was single-stranded facilitated the detection of the complex. Moscoso et al. (22) failed in directly detecting the covalent complex in the pMV158 system, but their study on the chirality of phosphorothioate in the strand transfer reaction indicated the existence of a covalent complex as a transient intermediate. Their conclusion on RepB protein of pMV158 agrees with our results on f1 gpII reported here.

   A

   B

   C

Figure 4. Interaction of Y197F gpII with the origin in vitro. (A) Gel retardation analysis of sequence-specific binding of gpII to the origin. Each lane contained 100 fmol of an origin-containing restriction fragment (origin DNA, 393 bp) and indicated amounts of the wild-type or mutant gpII. After incubation at 37°C for 15 min in a reaction buffer (20 mM Tris-Cl, pH 8.0, 1 mM EDTA, 5 mM DTT, 80 mM KCl, 5% glycerol), the samples were electrophoresed in a non-denaturing 5% polyacrylamide gel as described previously (14) and stained with ethidium bromide. Positions of complexes I and II, and of free origin DNA are shown on the left side. (B) Nicking assay using negatively supercoiled circular DNA (form I) as substrate. 0.3 pmol of form I plasmid DNA carrying the f1 origin was incubated with 3.2 pmol of gpII in a reaction buffer (20 mM Tris-Cl, pH 8.0, 5 mM MgCl2, 5 mM DTT, 80 mM KCl, 5% glycerol) at 37°C for 30 min, and electrophoresed on a 0.7% agarose gel containing ethidium bromide (7). (C) Nicking assay using synthetic oligonucleotide. 0.2 pmol of partially double-stranded substrate labeled at the 3[prime] end of the plus strand (Fig. 1A) was incubated with 2 pmol of gpII at 37°C for 5 min in the same buffer as in (B). Electrophoretic analysis was carried out as in Figure 1B. In each panel, lane 1 is a control without gpII.

gpII is a nicking-closing enzyme showing sequence-specific topoisomerase I-like activity (1,8). When incubated with wild-type gpII, ~60% of form I DNA molecules are converted to the nicked form (form II), while the remaining 40% are converted to relaxed closed form (form IV). No external energy source such as ATP is required for these reactions. The covalent complex reported here is most likely an intermediate in the nicking-closing reaction, and also in the nicking reaction. This mode of action of gpII is similar to that of DNA relaxases which play an essential role in the initiation and termination of conjugative DNA transfer in several plasmids, except for the remarkable difference in stability of the covalent nucleoprotein complex (reviewed in 24). Upon dissociation of the covalent bond, the phosphate group would be transferred to the 3[prime]-hydroxyl group of DNA in the nicking-closing reaction. Failure of the transfer to 3[prime]-OH would lead to interaction of phosphate with a water molecule, yielding nicked form DNA (form II). Such hydrolysis of phospho-tyrosyl bond between initiator protein and nicked DNA has been described for another rolling-circle plasmid replicon (25). A mutant gpII G73A gives a higher yield of covalent complex than wild-type gpII, suggesting that G73A mutation reduces the rate of dissociation of the covalent bond. We have previously shown that, upon incubation with f1 form I DNA, this mutant gpII produces more form II and less form IV compared to the wild-type (8). The decrease in the dissociation rate of the covalent bond seems to result in reduced joining and increased hydrolysis.

Does the gpII covalent complex play a role in DNA replication that follows the nicking? In [phis]X174, the stable covalent complex has been reported to facilitate the initiation of unwinding of duplex by rep helicase. The complex attaches to, and moves along with, the replication fork (26), forming a closed rolling circle intermediate where the end of the single-stranded tail is linked to the fork. In filamentous phage, on the other hand, a non-covalent complex consisted of gpII, and the origin has been shown to facilitate the initiation of unwinding (5). Rolling circle intermediates of filamentous phage appeared to have a single-stranded tail with a free end (5). These results suggest that, in filamentous phage, once the nick is introduced by the initiator, the presence of the covalent complex is not necessary for further steps of DNA replication. This notion is in accordance with rapid dissociation of the covalent complex we observed. However, all these data are from in vitro experiments and might not be representing the in vivo situation.

Our result directly shows that the covalent bond is formed between tyrosine 197 and the 5[prime] end of nicked DNA. Unlike the case of [phis]X174, where two tyrosine residues can form the covalent bond (27,28), only one tyrosine residue (Y197) was found covalently bound to DNA. Moreover, conversion of tyrosine 197 to phenylalanine abolished the in vivo activity to complement gene II amber mutation (Table 2). Purified preparation of Y197F gpII showed normal non-covalent binding to the origin and normal DNA bending, but failed to nick or relax the origin-containing form I DNA. These results clearly indicate that tyrosine 197 represents the active center of gpII nicking reaction, involving in the nucleophilic attack to the nick site of the origin.

Ilyina and Koonin (29) have identified three amino acid sequence motifs which are conserved among most initiator proteins for rolling circle type DNA replication. They are located in order from the N-terminus to the C-terminus of the protein. These ordered motifs were not found in filamentous phage gpII (29). It should be noted, however, that the amino acid sequence surrounding the tyrosine 197 residue of gpII (LVAYLKH) matches Ilyina-Koonin’s motif 3 (uxxYuxK/H, where u is a bulky hydrophobic residue, and x is any residue), which encompasses the tyrosine residue(s) that forms the covalent bond with nicked DNA. The seventh position of motif 3 is lysine in most initiator proteins, while it is histidine in gpII as well as in plasmids of the pMV158 group, where the covalent complex has not been detected. Replacement of this histidine in gpII by lysine did not make the covalent complex more stable (our unpublished data).

From the results reported here, it is possible that in all the rolling circle replication systems the nicking occurs through formation of covalent complex between initiator protein and nicked DNA. In most cases the covalent complex is stable, while it rapidly dissociates in filamentous phage and pMV158 type plasmids. The dissociation is catalyzed by the initiator protein itself. The biological significance of stability versus instability of the complex in terms of mode of DNA replication remains to be elucidated. In filamentous phage, instability of the complex may be related to the unique mode of regulated DNA replication which allows coordinated growth of the phage and the infected host cell (reviewed in 30).

ACKNOWLEDGEMENTS

This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan and the joint research program of the Institute of Genetic Ecology, Tohoku University.

REFERENCES

1. Meyer,T.F. and Geider,K. (1979) J. Biol. Chem., 254, 12642-12646. MEDLINE Abstract

2. Higashitani,A., Greenstein,D., Hirokawa,H., Asano,S. and Horiuchi,K. (1994) J. Mol. Biol., 237, 388-400. MEDLINE Abstract

3. Horiuchi,K. (1986) J. Mol. Biol., 188, 215-223. MEDLINE Abstract

4. Greenstein,D. and Horiuchi,K. (1987) J. Mol. Biol., 197, 157-174. MEDLINE Abstract

5. Geider,K., Baumel,I. and Meyer,T.F. (1982) J. Biol. Chem., 257, 6488-6493. MEDLINE Abstract

6. Harth,G., Baumel,I., Meyer,T.F. and Geider,K. (1981) Eur. J. Biochem, 119, 663-668. MEDLINE Abstract

7. Dotto,G.P., Horiuchi,K. and Zinder,N.D. (1984) J. Mol. Biol., 172, 507-512. MEDLINE Abstract

8. Higashitani,A., Greenstein,D. and Horiuchi,K. (1992) Nucleic Acids Res., 20, 2685-2691. MEDLINE Abstract

9. Ikeda,J.-E., Yudelevich,A., Shimamoto,N. and Hurwitz,J. (1979)J. Biol. Chem., 254, 9416-9428. MEDLINE Abstract

10. Eisenberg,S. and Kornberg,A. (1979) J. Biol. Chem., 254, 5328-5332. MEDLINE Abstract

11. Khan,S.A. (1997) Microbiol. Mol. Biol. Rev., 61, 442-455. MEDLINE Abstract

12. del Solar,G., Giraldo,R., Ruiz-Echevarria,M.J., Espinosa,M. and Diaz-Orejas,R. (1998) Microbiol. Mol. Rev., 62, 434-464.

13. Firth,N., Ippen-Ihler,K. and Skurray,R.A. (1996) In Neidhardt,F.C. (ed.), Escherichia coli and Salmonella typhimurium, 2nd Edn. American Society for Microbiology, Washington DC, pp. 2377-3401.

14. Greenstein,D. and Horiuchi,K. (1990) J. Mol. Biol., 211, 91-101. MEDLINE Abstract

15. Van Mansfeld,A.D.M., Van Teeffelen,H.A.A.M., Baas,P.D., Veeneman,G.H., Van Boom,J.H. and Jansz,H.S. (1984) FEBS Lett., 173, 351-356.

16. Ito,W., Ishiguro,H. and Kurosawa,Y. (1991) Gene, 102, 67-70. MEDLINE Abstract

17. Johnston,S. and Ray,D.S. (1984) J. Mol. Biol., 177, 685-700. MEDLINE Abstract

18. Greenstein,D., Zinder,N.D. and Horiuchi,K. (1988) Proc. Natl Acad. Sci. USA, 85, 6262-6266. MEDLINE Abstract

19. Dotto,G.P. and Zinder,N.D. (1984) Proc. Natl Acad. Sci. USA, 81, 1336-1340. MEDLINE Abstract

20. Dotto,G.P. and Zindr,N.D. (1984) Nature, 311, 279-280. MEDLINE Abstract

21. Kim,M.H. and Ray,D.S. (1985) J. Virol., 53, 871-878. MEDLINE Abstract

22. Moscoso,M., Eritja,R. and Espinosa,M. (1997) J. Mol. Biol., 268, 5222-5228.

23. Mizuuchi,K. and Adzuma,K. (1991) Cell, 66, 129-140. MEDLINE Abstract

24. Byrd,D.R. and Matson,S.W. (1997) Mol. Microbiol., 25, 1011-1022. MEDLINE Abstract

25. Noirot-Gros,M.F. and Ehrlich,S.D. (1996) Science, 274, 777-780. MEDLINE Abstract

26. Eisenberg,S., Griffith,J. and Kornberg,A. (1977) Proc. Natl Acad. Sci. USA, 74, 3198-3202. MEDLINE Abstract

27. Roth,M.J., Brown,D.R. and Hurwitz,J. (1984) J. Biol. Chem., 259, 10556-10568. MEDLINE Abstract

28. Van Mansfeld,A.D.M., Van Teeffelen,H.A.A.M., Baas,P.D. and Jansz,H.S. (1986) Nucleic Acids Res., 14, 4229-4238. MEDLINE Abstract

29. Ilyina,T.V. and Koonin,E. (1992) Nucleic Acids Res., 20, 3279-3285. MEDLINE Abstract

30. Model,P. and Russel,M. (1988) In Calendar,R. (ed.), The Bacteriophages, vol. 2. Plenum Press, New York, pp. 375-456.

31. Horiuchi,K. and Zinder,N.D. (1967) Science, 156, 1618-1623. MEDLINE Abstract

32. Sammbrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

33. Loeb,T. (1960) Science, 131, 932-933.

34. Lyond,B.L. and Zinder,N.D. (1972) Virology, 49, 45-60.

35. Fulford,N. and Model,P. (1988) J. Mol. Biol., 203, 49-62. MEDLINE Abstract

*To whom correspondence should be addressed at: Institute of Genetic Ecology, Tohoku University, Sendai 980-8577, Japan. Tel: +81 22 217 5715; Fax: +81 22 263 9845; Email: ahigashi@ige.tohoku.ac.jp
Present addresses: +Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan and §The Rockefeller University, Box 98, New York, NY 10021, USA

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 26 Mar 1999
Copyright©Oxford University Press, 1999.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Kuzminov
Single-strand interruptions in replicating chromosomes cause double-strand breaks
PNAS, July 17, 2001; 98(15): 8241 - 8246.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
S. Marsin, E. Marguet, and P. Forterre
Topoisomerase activity of the hyperthermophilic replication initiator protein Rep75
Nucleic Acids Res., June 1, 2000; 28(11): 2251 - 2255.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (220K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Asano, S.
Right arrow Articles by Horiuchi, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Asano, S.
Right arrow Articles by Horiuchi, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?