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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Replication termination system of B. subtilis
  5. Structure and function of the replication terminator protein (RTP) of B. subtilis
  6. Ter sequences of E. coli and R6K
  7. Structure and function of the Tus protein of E. coli
  8. Physiological significance of replication termini
  9. Acknowledgements
  10. References

Sequence-specific replication termini occur in many bacterial and plasmid chromosomes and consist of two components: a cis-acting ter site and a trans-acting replication terminator protein. The interaction of a terminator protein with the ter site creates a protein–DNA complex that arrests replication forks in a polar fashion by antagonizing the action of the replicative helicase (thereby exhibiting a contrahelicase activity). Terminator proteins also arrest RNA polymerases in a polar fashion. Passage of an RNA transcript through a terminus from the non-blocking direction abrogates replication termination function, a mechanism that is likely to be used in conditional termini or replication check points.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Replication termination system of B. subtilis
  5. Structure and function of the replication terminator protein (RTP) of B. subtilis
  6. Ter sequences of E. coli and R6K
  7. Structure and function of the Tus protein of E. coli
  8. Physiological significance of replication termini
  9. Acknowledgements
  10. References

In most bacterial and plasmid replicons, initiation of DNA replication occurs at specific origins and, during theta replication, the replication forks, moving unidirectionally or bidirectionally, are arrested at sequence-specific replication termini (for a review, see Bastia and Mohanty, 1996). The existence of specific replication termini was first discovered in plasmid R6K (Lovett et al., 1975; Crosa et al., 1976), and was subsequently found in the Gram-negative bacterium Escherichia coli (Kuempel et al., 1977; Louarn et al., 1977) and the Gram-positive bacterium Bacillus subtilis (Weiss and Wake, 1984; Smith and Wake, 1992). It should be noted that these specific replication termini do not effect termination during rolling circle DNA replication (Kaul et al., 1994). This review will consider recent developments in the study of termination of DNA replication, including the relatively sparse information on the physiological significance of specific replication termini.

Replication termini (ter ) are DNA sequences that interact with the replication terminator proteins and the ter–terminator protein complex arrests replication forks in a polar fashion. For a given orientation of the terminator, replication forks approaching from one direction are arrested, whereas replication forks from the opposite direction pass through unimpeded (Hill et al., 1988; Horiuchi and Hidaka, 1988; Sista et al., 1989; Smith and Wake, 1992). The replication terminator proteins of E. coli and B. subtilis are different in their three-dimensional structure, yet both proteins act similarly by arresting both replicative helicases (thereby possessing a contrahelicase activity) and RNA polymerases in a polar fashion (for a review, see Bastia and Mohanty, 1996).

Arrest of the replication forks at the termini constitutes the first step of termination. The orderly completion of the two circular daughter chromosomes without generating branched templates for rolling circle replication requires the termination system. This review will focus on the structure and function of the replication terminator proteins of B. subtilis and E. coli and the replication termini of bacterial and plasmid systems. This review will also consider the physiological roles of specific replication termini.

Replication termination system of B. subtilis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Replication termination system of B. subtilis
  5. Structure and function of the replication terminator protein (RTP) of B. subtilis
  6. Ter sequences of E. coli and R6K
  7. Structure and function of the Tus protein of E. coli
  8. Physiological significance of replication termini
  9. Acknowledgements
  10. References

The replication termini of B. subtilis are located in two clusters at a location ≈ 180° from the origin of replication (ori ); these are ordered in such a way that each cluster has opposite polarity and are therefore inverted repeats. Replication forks approaching from the counterclockwise direction pass through the first cluster of non-arresting termini and are arrested at the second set of termini which have the proper polarity (Fig. 1, A and B). Likewise, the clockwise forks pass through the right terminus and are arrested at the left terminus, with TerI (IRI) being the most frequently used terminus in vivo (for a review, see Wake and King, 1997). The Ter sequence is bipartite and overlapping, with each inverted repeat consisting of both a ‘core’ sequence (IRIB) and an ‘auxiliary’ (IRIA) sequence. The core sequence binds to a dimer of the replication termination protein (hereafter RTP), a sequence-specific DNA-binding protein, and this protein–DNA complex co-operatively allows the binding of a second dimer of RTP to the auxiliary site (Carrigan et al., 1991; Sahoo et al., 1995a). The auxiliary site does not bind to RTP in the absence of the core site, and two interacting dimers of RTP are needed to effect replication fork arrest (Sahoo et al., 1995a; Manna et al., 1996a). The interaction of RTP with the ter sequence causes an ≈ 30° bend in the DNA (Kralicek et al., 1997). However, the mechanistic significance of DNA bending in replication termination is not clear at the present time.

image

Figure 1. . The replication termini of plasmid R6K, E. coli and B. Subtilis. A. The arrangements of Ter sites at the replication termini of E. coli, B. subtilis and plasmid R6K. Note that the Ter sites are arranged in two sets of inverted repeats so as to form a replication trap. The dotted arrows show the direction of fork movement. B. The location of the replication check points (ψ) and the IRI and IRII replication termini of B. subtilis. The structure of the IRI sequence consisting of overlapping core (IRIB) and auxiliary (IRIA) sites is also shown; the direct repeats within the sites are boxed. C. A model which explains the mechanism of conditional arrest of forks at ψ sites by the sweeping away of RTP from ψ sites by transcriptional progression during the relaxed state, but not in the stringent state.

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In a naturally occurring plasmid of B. subtilis, a bipolar terminus has been discovered that arrests replication forks from both directions and in which each inverted repeat has two copies of the core sequence and lacks an auxiliary sequence (Meijer et al., 1996). This discovery suggests that the relative avidity of the interaction of RTP with the core is a major factor in determining polarity. The replication fork approaching the weaker auxiliary site is able to travel through unimpeded, whereas the fork approaching the core site (which has stronger affinity for the terminator protein) is arrested in a unipolar fashion. The bipolar terminus, in which both replication forks encounter a core site first, arrests the replication fork from both directions.

Structure and function of the replication terminator protein (RTP) of B. subtilis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Replication termination system of B. subtilis
  5. Structure and function of the replication terminator protein (RTP) of B. subtilis
  6. Ter sequences of E. coli and R6K
  7. Structure and function of the Tus protein of E. coli
  8. Physiological significance of replication termini
  9. Acknowledgements
  10. References

RTP is a homodimer of subunit molecular weight of 14 500 Da. The structure of the apoprotein has been solved at 2.6 Å (Bussiere et al., 1995; Fig. 2A). The protein belongs to the ‘winged helix’ family of proteins and consists of four α-helices, three β-strands, an extended loop and an unstructured, flexible, N-terminal arm; the strands and loop form the ‘wing’ of the winged helix (Bussiere et al., 1995). The α4 helix forms a rather large antiparallel coiled coil with its symmetry mate in the homodimer, and this serves as the major determinant of dimerization. Initial model building and analysis of the electrostatic potential of the protein suggested that the α3 helix and β2 strand were the most likely to contact DNA, with α3 being the major groove-binding DNA-recognition helix (Bussiere et al., 1995). Analysis of the structure against the current structures in the Protein Data Bank reveals that the closest structural homologue to RTP is the histone H5 protein, although it is also similar to other winged helix proteins (PDB accession code 1hst-1). This homology derives from the structural similarity between the DNA-binding regions (the winged helix) and suggests that RTP evolved from an archetypal DNA-binding protein. It should be noted that there is no significant sequence similarity between RTP and the histone H5 protein.

image

Figure 2. . Structural diagrams of RTP. A. Ribbon diagram of a monomer showing the four α-helices, three β-strands, an extended loop connecting β2 and β3, and the unstructured N-terminal arm. The structure is a member of the winged helix family of proteins, with the wing being formed by the three β-strands. B. The exposed hydrophobic patch of RTP. Mutations at Glu-30 and Tyr-33 cause partial and complete loss of contrahelicase activity with respect to the DnaB helicase of E. coli, without detectable loss of DNA binding, proper protein folding, dimerization, or dimer–dimer interaction. The mutations reduce in vitro interaction between DnaB and RTP. This region has been proposed as the contrahelicase surface of RTP. C. A model showing the interaction between two RTP dimers in the region of the ‘wing’; upon mutation to other residues, residues Gly-34 (G34), Tyr-88 (Y88), Leu-82 (L82) and Val-85 (V85) abolish dimer–dimer interaction, but not proper protein folding, dimerization or DNA binding.

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Random and structure-guided rational mutagenesis, purification of the mutant forms, and their biochemical analysis has revealed the various functional regions of the protein (Manna et al., 1996a; 1996b; Pai et al., 1996a). The regions of the protein that contact DNA were identified by a selection scheme designed for isolation of a DNA-binding defect from a population of random mutants. This procedure implicated the unstructured N-terminal arm and α3 in DNA binding. The mutational results were confirmed by site-directed azido cross-linking (Pai et al., 1996a,b). The contact points of individual amino acid residues to bases were derived by selectively coupling the individual residues with Fe-EDTA and binding the derivatized RTP to DNA, followed by inducing cleavage at or near the contact points by providing H2O2 to promote the generation of hydroxy radicals that cleave DNA. By using a novel computational algorithm (based on the distance geometry method used in macromolecular NMR) that uses the distance between the amino acid and the base, as derived from the length of the Fe-EDTA adduct and the quenching distance of the hydroxy radical, an experimental model of the RTP–DNA protein complex was derived (Pai et al., 1996b; Fig. 3B, top). Thus, conversion of RTP into a site-directed chemical nuclease confirmed that the α-3 helix, the β-2 strand and the N-terminal arm were the regions of RTP that contacted DNA. This is shown in red in 3Fig. 3A.

image

Figure 3. . Comparative structures of the replication terminator proteins of B. subtilis and E. coli. A. Crystal structure of RTP (Bussiere et al., 1995); the identified DNA-binding (red), dimer–dimer interaction (blue), and contrahelicase (yellow) regions are shown. B. A model showing the RTP–Ter complex in which the helicase approaching the auxiliary site displaces the RTP and passes through unimpeded, whereas the helicase approaching the core site is arrested (Bussiere et al., 1995; Pai et al., 1996b); DNA-binding (red), dimer–dimer interaction (blue), and contrahelicase (yellow) regions are shown. C. Crystal structure of the Tus–Ter complex of E coli (Kamada et al., 1996) showing the DNA-binding region of β-strands and the helicase-blocking end.

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The crystal structure of RTP revealed an exposed hydrophobic patch on RTP that was suggested as a possible protein interaction surface for the helicase (Fig. 2B). Extensive mutagenesis studies have revealed that mutations at the Glu-30 and Tyr-33 residues do not detectably affect the dimerization, DNA binding, or dimer–dimer interaction properties of the protein. However, in every single mutant of Glu-30 (four out of four), the contrahelicase activity is significantly diminished in vitro at a ratio of 20-fold RTP over the DNA substrate (Manna et al., 1996b; D. Bastia, in preparation). The ability of the mutant forms of the protein to arrest replication forks in vitro is correspondingly reduced.

Mutations at the Tyr-33 residue almost completely abolish contrahelicase activity without detectably impairing the DNA-binding activity, dimer–dimer interaction, or dimerization properties of the protein. The physical integrity of the mutant forms of RTP have been carefully established by X-ray crystallography. In vitro binding studies also revealed that mutations at Glu-30 and Tyr-33 residues correspondingly reduce the protein–protein interaction between the DnaB helicase, the major replicative helicase in the cell, and RTP (Manna et al., 1996b; Fig. 2B). The contrahelicase activity recognizes several replicative helicases, but not the Rep helicase of E. coli or helicase I (F plasmid TraI protein) or helicase II (E. coli uvrD), and therefore has some specificity (Sahoo et al., 1995b). RTP does not arrest rolling circle replication in vitro (Kaul et al., 1994) or in vivo (Bruand et al., 1990), a fact that is consistent with its inability to arrest the Rep helicase of E. coli which is known to be involved in rolling circle replication of phage φx174 (Sahoo et al., 1995b).

The dimer–dimer interaction surface of RTP has been identified by X-ray crystallography and site-directed mutagenesis (Manna et al., 1996a). The data firmly implicate the β-3 strand and the tip of the extended loop in the interaction. A model of an interacting pair of RTP dimers is shown in 2Fig. 2C (Manna et al., 1996a)

On the basis of the crystal structure, mutagenesis and biochemical analysis, the data best fit the model shown in 3Fig. 3B. The avidity of binding of RTP to the ter site is most probably the primary determinant of polarity. For helicase arrest to occur, however, both RTP–DNA interaction and RTP–helicase contacts seem to be necessary. The helicase putatively contacts the RTP at the contrahelicase surface. Either the Glu-30 or Tyr-33 residues play a direct role in promoting the helicase–contrahelicase contact, or mutation at these residues alters the contrahelicase surface in such a way as to reduce the interaction. The replication fork approaching the auxiliary site would displace the dimer binding to that site by reducing the overall co-operativity of the RTP dimer–dimer interaction and sweeping through the terminus. Forks approaching the core site are unable to displace the protein (Fig. 3B). Whether the different affinities of RTP for the core and auxiliary sites is the only determinant of polarity, or if the structure of the protein–DNA complex at the core site is slightly different from that at the auxiliary site, thus contributing to the functional asymmetry with a structural asymmetry, will have to await the determination of the crystal structure of the ter–RTP complex. Co-crystals of the RTP–IRI complex have been obtained and are currently being optimized for subsequent structural solution (M. Swan, C. Davies and S. W. White, unpublished).

Ter sequences of E. coli and R6K

  1. Top of page
  2. Abstract
  3. Introduction
  4. Replication termination system of B. subtilis
  5. Structure and function of the replication terminator protein (RTP) of B. subtilis
  6. Ter sequences of E. coli and R6K
  7. Structure and function of the Tus protein of E. coli
  8. Physiological significance of replication termini
  9. Acknowledgements
  10. References

Sequence-specific replication termini were first discovered in the plasmid R6K (Lovett et al., 1975; Crosa et al., 1976). The first identification and nucleotide sequence of a ter region and the development of the first in vitro system to analyse replication termination was also carried out in R6K (Bastia et al., 1981a; 1981b; Germino and Bastia, 1981). Subsequent work narrowed down the minimal sequence of ter from 216 bp to ≈ 20 bp (Horiuchi and Hidaka, 1988). The ter sequences of plasmid R6K and the host E. coli are shown in 1Fig. 1A. The ter sites of E. coli and R6K, as in B. subtilis, are again arranged as two groups of opposite polarity (inverted repeats). Unlike the ter sites of B. subtilis, however, the sites of E. coli are not bipartite, and each site binds to a single monomer of Tus protein, the DNA replication terminator protein in E. coli, which has a molecular weight of 36 000 Da (Hill et al., 1989; Sista et al., 1989). As in the case of the RTP–DNA complex, the Tus–DNA complex acts as a polar contrahelicase of replicative helicases (Khatri et al., 1989; Lee et al., 1989; Hiasa and Marians, 1992), and it also arrests several RNA polymerases in a polar fashion (Mohanty et al., 1996; 1998).

The replication termini of R6K are located asymmetrically with respect to the origin of replication. Using a clock analogy, keeping the two origins of replication at ≈ 12 o'clock, the pair of ter sites are located at the 2 o'clock position (Lovett et al., 1975; Crosa et al., 1976). The presence of the replication termini significantly affects the topology of replication and makes it sequentially bidirectional. Thus, one of the three origins (designated α, β and γ) initiate replication in any given chromosome by generating a unidirectionally moving replication fork that is arrested upon reaching ter. The origin then initiates a fork moving in the opposite direction that meets and merges with the first arrested fork after replicating the rest of the chromosome, thereby imparting a sequentially bidirectional mode of replication (Lovett et al., 1975; Crosa et al., 1976). In the absence of ter, the replication becomes unidirectional (Crosa et al., 1978). The ter sites of E. coli are shown in 1Fig. 1A (Hill et al., 1988).

Structure and function of the Tus protein of E. coli

  1. Top of page
  2. Abstract
  3. Introduction
  4. Replication termination system of B. subtilis
  5. Structure and function of the replication terminator protein (RTP) of B. subtilis
  6. Ter sequences of E. coli and R6K
  7. Structure and function of the Tus protein of E. coli
  8. Physiological significance of replication termini
  9. Acknowledgements
  10. References

The crystal structure of the ter–Tus complex has been solved at 2.7 Å (Kamada et al., 1996; Fig. 3C). Unlike RTP, which is a winged helix protein, the Tus protein has a unique overall fold with a set of β-strands that invade the major groove of ter DNA. The helicase-arresting end projects over the DNA-binding domain (Fig. 3C). The Tus protein is structurally unrelated to other known protein structures, most notably RTP. Most of the mutants that abolish contrahelicase activity have been located at the DNA-binding domain. The Tus protein has not been analysed by mutagenesis as thoroughly as RTP, presumably because the former is a larger protein. There is in vitro evidence showing interaction between DnaB and Tus protein, but the specificity and biological relevance of this interaction remains undetermined (B. K. Mohanty and D. Bastia, unpublished). Tus protein also arrests RNA polymerases in a polar fashion (Mohanty et al., 1996; 1998). The permissive (non-blocking) ends of the ter sites are protected by sequences that are structurally similar to ρ-independent transcription terminators; these sequences presumably protect the sites from inactivation by transcriptional invasion (Mohanty et al., 1996).

Physiological significance of replication termini

  1. Top of page
  2. Abstract
  3. Introduction
  4. Replication termination system of B. subtilis
  5. Structure and function of the replication terminator protein (RTP) of B. subtilis
  6. Ter sequences of E. coli and R6K
  7. Structure and function of the Tus protein of E. coli
  8. Physiological significance of replication termini
  9. Acknowledgements
  10. References

There seems to be no causal relationship between the termination of a round of replication and subsequent cell division (Dasgupta et al., 1992). However, it is common knowledge that very few cells are generated during cell division that lack or have an incomplete copy of the chromosome. Therefore, the process of DNA replication must somehow be co-ordinated with cell division (Dasgupta et al., 1992). In plasmid R100, inactivation of the replication terminus leads to plasmid instability and the generation of rolling circles. The ter system seems to prevent the 3′ end of the leading strand of replicating DNA from displacing the 5′ end of the strand, thus generating the template for a rolling circle (Krabbe et al., 1997; Fig. 4B, bottom). It is also difficult to speculate as to why each chromosome has several termini, rather than the minimal requirement of two (one to stop the clockwise replication fork and the other to stop the counter-clockwise replication fork). It is possible that because the ter–terminator protein complex is not capable of holding back the replication fork indefinitely the multiple termini are needed to effectively co-ordinate fork arrest during a round of replication.

image

Figure 4. . Termination of replication after fork arrest at Ter sites. A. (1) Melting of the DNA helix and repair synthesis generates a catenated dimer (2), that is decatenated (3) by the activity of topoisomerase IV (Zechiedrich and Cozzarelli, 1995). B. In the absence of a functional terminus, rolling circles are generated at the termination stage, thus leading to chromosome instability (Krabbe et al., 1997).

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In B. subtilis, stringent conditions induce the gene relA that encodes ppGpp synthetase, which in turn elevates the intracellular concentrations of the alarmone ppGpp. Although under relaxed conditions in B. subtilis replication forks move unimpeded until reaching the ter sites, under stringent conditions the forks are arrested at two sites (called ψ, Fig. 1B) that are located ≈ 200 kb on either side of the origin of replication. The arrest at the ψ sites also requires RTP (Levine et al., 1991; 1995). In principle, several possible mechanisms can be invoked to explain the arrest of forks at the ψ check points. First, RTP could be modified under stringent conditions and this modification could allow it to recognize the ψ site. Second, another protein could be induced that helps RTP bind to the ψ site under stringent conditions. Third, transcriptional alterations brought about by high levels of ppGpp in the cell might alter the topology of the chromosome, thereby allowing RTP to bind to ψ sites. Fourth, consistent with the observation that transcriptional passage abrogates termination of replication (Mohanty et al., 1996; 1998) and that a large number of transcripts are turned off by the interaction of ppGpp with RNA polymerase (Cashel et al., 1996), ψ sites could be rendered non-functional by transcriptional passage during the relaxed state. Upon elevation of the concentrations of the alarmone, ppGpp, in the cell, the transcript could be turned off, thus leading to binding of RTP to ψ sites and conditional fork arrest (Fig. 1C). Recent work from our laboratory is consistent with the last model (A. Gautam, B. K. Mohanty and D. Bastia, in preparation).

The arrest of the replication fork is only one of the steps of replication termination. After fork arrest, a two-step process involving the melting of the DNA helix and subsequent repair synthesis leads to an intermediate of two catenated daughter molecules that can be decatenated by topoisomerase IV (Zechiedrich and Cozzarelli, 1995; Fig. 4A). Also, a system of a specific recombination site and the Xer recombinase acts to resolve oligomers if an odd number of cross-overs between the two daughter molecules generate oligomeric DNA (Blakely et al., 1991; Kuempel et al., 1991).

A critical question to be answered is how divergent are termination systems from one another? Given the dissimilarity between the termination systems of E. coli and B. subtilis (i.e. the use of structurally disparate terminator proteins and termini), will each organism in which a termination system is discovered use a unique set of proteins and termini, or will the systems be able to be grouped into related classes? It is also likely that the site-specific termination systems serve other physiological functions and future work should be directed towards unravelling these roles and to investigate site-directed termination systems in eukaryotic chromosomal and mitochondrial DNAs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Replication termination system of B. subtilis
  5. Structure and function of the replication terminator protein (RTP) of B. subtilis
  6. Ter sequences of E. coli and R6K
  7. Structure and function of the Tus protein of E. coli
  8. Physiological significance of replication termini
  9. Acknowledgements
  10. References

This work was supported by a Merit award from NIAID to D.B. and a grant from NIGMS to D.B. and Stephen W. White. We would like to thank Dr Stephen White (St. Jude Children's Research Hospital) for early communication of results and for helpful discussions.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Replication termination system of B. subtilis
  5. Structure and function of the replication terminator protein (RTP) of B. subtilis
  6. Ter sequences of E. coli and R6K
  7. Structure and function of the Tus protein of E. coli
  8. Physiological significance of replication termini
  9. Acknowledgements
  10. References
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