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Primosome Assembly Site in Bacillus Subtilis

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Primosome Assembly Site in Bacillus Subtilis The EMBO Journal vol.14 no.11 pp.2642-2650, 1995 Primosome assembly site in Bacillus subtilis Claude Bruand1, S.Dusko Ehrlich and involving the Rep protein, was proposed (Bruand et al., Laurent Janniere 1993). In contrast, little is known about the requirements for lagging strand synthesis of pAMf1-related plasmids. Laboratoire de Genetique Microbienne, Institut de Biotechnologie, It was reported that it may depend on the dnaD gene in INRA - Domaine de Vilvert, 78352 Jouy en Josas Cedex, France Bacillus subtilis (Ceglowski et al., 1993), but the function 'Corresponding author of this gene is unknown. Initiation of lagging strand synthesis is well documented A single-strand initiation site was detected on the in rolling circle replicating genomes such as single stranded Enterococcus faecalis plasmid pAMP1 by its ability to (ss) DNA phages and ss DNA plasmids. In these replicons, prevent accumulation of single stranded DNA of a lagging strand synthesis initiates at an origin called sso rolling circle plasmid, both in Bacillus subtilis and (for single-strand origin; Baas and Jansz 1988; Gruss and Staphylococcus aureus. This site, designated ssiA, is Ehrlich 1989). The sso belong to the family of single- located on the lagging strand template, -150 bp down- strand initiation sites (ssi), defined as sequences which stream from the replication origin. ssiA priming activity direct primer synthesis on ss DNA templates, either by requires the DnaE primase, the DnaC replication fork RNA polymerase or by a DNA primase. ssi were also helicase, as well as the of the dnaD products dnaB, and detected in certain theta replicating plasmids. In some dnal genes of B.subtilis, but not the RNA polymerase. these sites were shown to be involved in The primase and the instances, the replication fork helicase require- priming of either leading or lagging strand DNA synthesis ments indicate that ssiA is a primosome assembly site. (Minden and Marians, 1985; Masai and Arai, 1989b; Interestingly, the pAM01 lagging strand synthesis is Masai etal., 1990; Honda etal., 1992; Kubotaetal., 1993). inefficient when any of the proteins involved in ssiA In all ssi activity is but occurs in Gram-positive bacteria, found so far originate mutated, efficiently the absence from rolling circle plasmids, and the majority of them of ssiA. This suggests that normal plasmid replication RNA requires primosome assembly and that the primosome require polymerase. Here we describe a new type can assemble at of ssi, present in the theta replicating plasmid pAMP1. not only ssiA but also elsewhere on This is the plasmid. This work for the first time describes a site, designated ssiA, located -150 bp downstream primosome in a Gram-positive bacterium. Involvement of the replication origin, on the lagging strand template. of the B.subtilis proteins DnaB, DnaD and DnaI, which ssiA activity is dependent on DnaE primase, DnaC helicase do not have any known analogue in Escherichia coli, and the products of the dnaB, dnaD and dnaI genes of raises the possibility that primosome assembly and/or B.subtilis, but not on RNA polymerase. Primase and function in B.subtilis differs from that in E.coli. helicase requirements indicate that ssiA is a primosome Key words: Bacillus subtilislDNA replication/helicase/ assembly site. Requirement for proteins without known primase/primosome assembly site homologue in E.coli (the B.subtilis DnaB, DnaD and DnaI) suggests that primosome assembly and/or function in B.subtilis differ from those in E.coli or that the E.coli analogues exist but have not yet been identified. Proteins Introduction involved in ssiA activity are required for efficient lagging strand synthesis of plasmids derived from pAMP1, which pAMP1 is a 26.5 kb plasmid isolated from Enterococcus suggests that assembly of a primosome is necessary for faecalis (Clewell et al., 1974). It belongs to a large family normal pAM,B1 replication. of closely related, broad host range plasmids, which includes pIP501 from Streptococcus agalactiae and pSM 19035 from Streptococcus pyogenes (see Janniere Results et al., 1993 for a review). Replication of these plasmids is dependent on a plasmid encoded protein (Rep) and Detection and mapping of ssiA proceeds by a unidirectional theta mechanism, initiated In order to detect a ssi on pAM31 we made use of plasmid downstream of the rep gene, within an origin of <50 bp pHV1610 (Boe et al., 1989). This plasmid is a hybrid (Figure lA; Swinfield et al., 1990; Bruand et al., 1990, between pC194 and pUC19 (Figure IC) and replicates in 1991, 1993; Brantl and Behnke 1992a,b; Ceglowski et al., B.subtilis by a rolling circle mechanism, using the pC194 1993; Le Chatelier et al., 1993; E.Le Chatelier, personal replicon (Figure IB; Gros et al., 1987). The sso of pC 194 communication). Leading strand synthesis, which has been is inactive in this host and the plasmid accumulates ss studied in some detail, is initiated by DNA polymerase I DNA (Gruss et al., 1987). Insertion of an active ssi into (Pol I), a property in common with Escherichia coli pHV1610 reduces this accumulation, in an orientation- plasmids of the ColEl and ColE2 families. A model of specific manner (Figure IB; Boe et al., 1989). The plasmid initiation, based on that of ColE l-type plasmids but carries a convenient multiple cloning site (MCS), and 2642 K Oxford University Press ssiA of plasmid pAM,B1 A D repE ssiA 0.2 kb B C pHV1 61 0-1 5, pHVl 61 0-2 Ap 44 'dso pC194 x I ori pUC repA Fig. 1. (A) Schematic representation of a 2.8 kb HpaI-EcoRI segment of pAMPl, present in plasmid pIL253, which contains the minimal replicon. Open reading frames are represented by arrows and the replication origin (ori) and direction of replication by an open triangle. ssiA is represented as a black box. (B) Description of rolling circle replication. A strand-specific nick is introduced by the plasmid replication protein in the double-strand origin (dso, indicated by a bent arrow). The 3' end at the nick is used as a primer for leading strand elongation, while the lagging strand template is displaced. After one round of replication, the replication protein introduces a second nick at the same site in ori, and ligates the DNA ends, producing a ds and a ss DNA molecule. The ss DNA is then converted to a ds form. This step is stimulated if the ss DNA molecule carries a ssi, represented by a black box. (C) Schematic representation of plasmid pHV1610 and its derivatives pHVI610-1 and 2. Symbols are as in (A) and (B). replicates in E.coli, which facilitates construction of 1610-1 1610-2 1610 different derivatives. A segment of pAM,1 encompassing 57 bp of the 3' W,,* XF9Mw ...... end of the repE gene, the replication origin and -450 bp of the downstream region (Figure IA), was inserted in both orientations into the MCS of pHV1610. The resulting HMW plasmids, pHVI610-1 and pHVI610-2 (Figure IC), were introduced into the B.subtilis strain 1012 and tested for ss DNA accumulation. The amount of ss DNA of pHV1610-2 was similar to that of the parental plasmid, whereas that of pHV 1610-1 was much lower (Figure 2, two independent transformants were tested for each construct). These results -m %P. -o -m suggest that pAM,1 carries a ssi, designated ssiA, on the ccc lagging strand template. To map ssiA more precisely, the effect of different short pAM, segments on ss DNA accumulation was tested as above. The results, summarized in Figure 3A, show that SS --tom s ssiA is active in plasmids Al and AD5.18 but not in plasmids AG3.8 and AD5.24. The sequence of the relevant pAM,B1 region and the end-points of the segments used are shown in Figure 3B. ssiA is located in a 145 bp Fig. 2. Detection of ssiA. Bacillus subtilis 1012 cells harbouring segment (coordinates 4712-4856 of to plasmids pHV1610, pHV1610-1 or pHV1610-2 were grown to mid-log pAMPI according phase and their total DNA was extracted and analysed by Southern Swinfield et al., 1990), which can form a hairpin structure. hybridization, using 32P-labelled pHV1610 DNA as probe. For pHVI610-1 and pHV1610-2, two independent constructs were tested. ssiA activity does not require RNA polymerase CCC, covalently closed circular monomer; ss, single stranded DNA; HMW, high molecular weight plasmid which accumulate in Most ssi described so far in Gram-positive bacteria require multimers, B.subtilis when pUC sequences are joined to rolling circle plasmids the host RNA polymerase. To test whether ssiA activity (Gruss and Ehrlich, 1988). also requires this enzyme, we determined the effect of rifampicin, a specific RNA polymerase inhibitor, on the accumulation of pHV1610-1 ss DNA. For this purpose on RNA polymerase (pHV1611, Boe et al., 1989), was B.subtilis 1012 cells harbouring the plasmid were grown used as control. pHV1610-1 did not accumulate ss DNA to mid-log phase, and rifampicin was added. The amount upon addition of rifampicin, even after prolonged incuba- of ss DNA was determined in samples withdrawn at tion, whereas pHV1611 did, as early as 10 min after different times after drug addition. A pHV1610 derivative addition of the drug (Figure 4). These results indicate that carrying the ssoU of plasmid pUB 110, which is dependent ssiA activity does not depend on RNA polymerase. 2643 C.Bruand, S.D.Ehrlich and L.Janniere p,HVI611 pHV1610-1 A ssi Nhel AseI EcoRI activity 0 10 20 30 60 0 10 20 30 60 p6or ssiA pHVl1610-1 D~~ 1+ Al + AG3.8 AD5.18 + AL Aik AIL AD5.24 ccc Vow 0.1 kb B G AA A G A.T G G T.
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