Roles of phi X174 type primosome- and G4 type primase-dependent primings in initiation of lagging and leading strand syntheses of DNA replication.

Eleven single strand initiation sequences (ssi) were isolated from various plasmid genomes using a plaque-morphology assay. Out of seven ssi that require dnaB and dnaC functions for replication in a crude in vitro system, six use a phi X174 type priming mechanism, and a phi X174 type primosome is assembled at these sequences from the purified proteins, n'(priA), n(priB), n"(priC), dnaT, dnaB, dnaC, and primase. The same ssi potentiate dATPase activity of n' protein, and thus represent new n' protein recognition sequences (n'-pas). Based on sequence homology, two structural groups are evident. Two sequences show a strong homology with the phi X174 site, whereas three share extensive homology with the previously characterized n'-pas of ColE1, ssiA(ColE1). All the n'-pas have a potential to form stem and loop structures, although sequence homology between the two classes is absent. In addition to the phi X174 type priming, three ssi do not require either dnaB or dnaC function for replication, and use a G4 type priming, requiring only SSB and primase. The 5' ends of primer RNA synthesized by primase are localized within the vicinity of one of the three blocks of highly conserved nucleotide sequences. Deletions of parts of these conserved sequences result in loss of priming activity, suggesting that they are important for priming on the G4 type ssi, which are termed G site. The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed.


Eleven
single strand initiation sequences (ssi) were isolated from various plasmid genomes using a plaquemorphology assay. Out of seven ssi that require dnaB and dnaC functions for replication in a crude in vitro system, six use a 4X174 type priming mechanism, and a 4X174 type primosome is assembled at these sequences from the purified proteins, n'(priA), n(priB), n"(priC), dnaT, dnaB, dntiC, and primase. The same ssi potentiate dATPase activity of n' protein, and thus represent new n' protein recognition sequences (n'pas). Based on sequence homology, two structural groups are evident.
Two sequences show a strong homology with the 4X174 site, whereas three share extensive homology with the previously characterized n'pas of ColEl, ssiA(ColE1). All the n'-pas have a potential to form stem and loop structures, although sequence homology between the two classes is absent. In addition to the 9X174 type priming, three ssi do not require either dnaB or dnaC function for replication, and use a G4 type priming, requiring only SSB and primase.
The 5' ends of primer RNA synthesized by primase are localized within the vicinity of one of the three blocks of highly conserved nucleotide sequences. Deletions of parts of these conserved sequences result in loss of priming activity, suggesting that they are important for priming on the G4 type ssi, which are termed G site. The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed.
In both prokaryotic and eukaryotic replicons, DNA chain elongation is generally initiated upon primer RNAs synthesized by primase or by RNA polymerase (1) revealed four different modes of priming (1,2). (i) Host RNA polymerase synthesizes an RNA primer at a defined region of SSB-coated Ml3 SS DNA (3). (ii) On SSB-coated G4 phage DNA, DNA primase (dnaG gene product) synthesizes an RNA primer at the complementary origin (4). (iii) Primer RNA synthesis on SSB-coated 4X174 DNA requires the action of a set of seven prepriming proteins for primase to recognize priming signals (5). A specific sequence, called the n/-pas,' is required for the assembly of a prepriming protein complex (6). Primer RNAs are synthesized at multiple sites located throughout the $X174 genome due to the mobile nature of this protein complex (7,8). (iv) Primase can also catalyze primer RNA synthesis on uncoated SS DNA in the presence of dnaB protein, without apparent sequence specificity (general priming) (9). In order to determine that priming signals like those found on SS phages are present on duplex genomes, we have been searching for single strand initiation sequences (ssi) based on their ability to complement the poor growth of a SS phage lacking its complementary origin (10, 11). ssi have been isolated from ColEl (12, 13), pBR322 (14), the f5 fragment of the F factor (15), RSFlOlO (16), and an Rl plasmid derivative (17). Two ssi from ColEl plasmid (ssiA and ssiB, identical to pas-BL and pas-BH, respectively, isolated from pBR322) and one from the F-f5 fragment (F ssiA) were shown to convert SS to RF in vitro by a 4X174 type mechanism (13-15), and thus represent n'-pas. Further analysis of the function of ssiA(ColEl), located on the lagging strand template downstream of ori, demonstrated the crucial role of the primosome assembled at this n'-pas in efficient lagging strand synthesis in vitro (l&19).
Furthermore, the unique site of leading strand initiation in RlOO and Rl plasmid replication maps at the region identical to one of the ssi isolated by the plaque assays (20,21 names will be reported elsewhere. In this report, we describe the mechanisms by which they promote SS to RF DNA conversion in uitro. Eleven newly isolated ssi could be divided into two major groups, those that direct the assembly of the 4x174 type primosome (n'-pas) and those that are recognized by primase directly (G site). Based on their locations on the genome relative to known replication origins and direction of replication, we propose the function of some of the ssi in priming leading or lagging strand synthesis. Comparison of n'-pas and G sites, as well as deletion analysis of G sites, provides some insight into how these sequences are recognized by the primosome and primase.

Isolation
of 11 ssi from Plasmid Replicons-By using a plaque-morphology assay (10, ll), we have isolated 11 ssi from F, R6K, RlOO, and ColE2 plasmids. Six are from the entire F plasmid genome, two from the R6K origin region, two from the basic replicon of RlOO, and one from the region down- Mechanisms of Priming in DNA Replication stream of the ColE2 origin. The maximal limit sizes of each ssi, delineated by in vivo plaque assays of deletion derivatives, are summarized in Table I. The precise locations and nucleotide sequences of these ssi will be reported elsewhere.
In order to elucidate the mechanisms of priming on these ssi, the recombinant phage DNAs described above were tested in in vitro replication assays. Crude fraction II extracts were prepared from dnaB and dnaC temperature-sensitive strains and SS to RF DNA replication was measured in the presence of rifampicin which suppressed RNA polymerase-dependent priming that may result from the vector. Requirement for dnaB and dnaC function was obvious in replication of ssiB(F.f2a), ssiC(F.f2b), ssiA(F.f5), ssiF(F.fl), ssiA(RGK), ssiB(RlOO), and ssiA(ColE2) ( Table I). ssiE(F. f6), ssiB(RGK), and ssiA(RlOO), on the other hand, did not require either dnaB or dnaC function for replication (Table I). Replication activity of ssiD(F. f3) was considerably lower than that of others in the fraction II system. However, in the absence of rifampicin, substantial replication was observed with ssiD(F. f3) (data not shown), suggesting the possibility that RNA polymerase may be responsible for the priming. It is interesting to note that those ssi that require dnaB and dnaC functions could be confined within some 100 bases, whereas others that are independent of dnaB and dnaC needed at least 200 bases for clear plaque formation in vivo. Six ssi Promote SS to RF DNA Replication by a 4X174 Type Mechanism-To examine whether those ssi requiring dnaB and dnaC for replication function to assemble a 4X174 type primosome, the template activities of the chimeric phage SS to RF replication of recombinant phage DNAs carrying ssi in the reconstituted in vitro 4x174 replication system SS to RF DNA replication was assayed, 10 min at 30 "C, in the in uitro 4X174 replication system reconstituted with purified proteins as described under "Materials and Methods" with 0. DNAs were tested for the ability to replicate in a purified system for 4X174 DNA synthesis. Among those templates requiring dnaB and dnaC activity in vitro, all but ssiA(R6K) were active in the reconstituted system (Table II). The prepriming proteins, n', n, nn, and dnaT, were required for replication of these six chimeric phage DNAs (Table III). The same set of templates served as an effector DNA of dATPase of n' protein, which triggers the assembly of the primosome (27) ( Table IV). These results indicate that ssiB(F'f?Za), ssiC(F.f2b), ssiA(F.f5), ssiF(F.ff), ssiB(RlOO), and ssiA (ColE2) promote priming by a 4x174 type mechanism. They are thus new isolates of n/-pas. Although ssiE(F. f6), ssiB(RGK), and ssiA(RlOO), which did not require either dnaB or dnaC in a crude system, were replicated to a significant extent in the reconstituted system (Table II), they did not require any of the prepriming proteins (data not shown, see below). ssiD(F.f3) was inactive in the reconstituted system. ssiA(RGK), which required both dnaB and dnaC, was neither replicated in the 1$X174 reconstitution system nor stimulated the dATPase activity of n' protein (Tables II and  IV), suggesting that the mechanism of priming on ssiA(R6K) differs from that of @X174. To simplify the following discussion, all the ssi replicating through a 4x174 type priming will be renamed as shown in Table IV. Two ssi, ssiA(ColE1) and ssiB(ColES), will be renamed n'-pasA(ColE1) and n'-pasB(ColEl), respectively. Sequence Homologies Among Various n'-pas-Besides the n'-pas of 4X174 DNA, three distinct n'-pas derived from plasmid DNAs had been previously described (n'-pasA and n'-pasB of ColEl and its related plasmids, and F ssiA of f5 fragment of F plasmid) (12-15). Identification of five additional novel n'-pas (n'-pas(F.f5) is identical to F ssiA previously reported (15)) enabled us to search for possible motifs recognized by n' protein. The n/-pas can be divided into at least two groups based on sequence homology (Fig. 1). Group I shows extensive sequence homology to the n'-pas of @X174, whereas group II is homologous to n'-pasA(ColE1).
n'-pas(F. f2b) and n'-pas(R1OO) belong to group I, while n'-pas(F.f5), n'-pas(F.ff), and n'-pas(ColE2) belong to group II. n  shows significant homology to the consensus sequences of group II, although some deviation is observed. n'-pas(F.f2a) shows only a limited homology to parts of 11 and 10 base stretches of the conserved sequences of group II (data not shown).
The n'-pas of 4X174, as well as n'-pasB(ColE1) (identical to pasBH of pBR322), are known to form a secondary structure resistant to digestion by exonuclease VII (6,28). Hairpin structures similar to the 4x174 n'-pas can be drawn for other group I ssi, since most of the bases participating in the stem  fall into the conserved sequences (Fig. 2). Hairpin structures can also be drawn for the members of group II, although thermodynamic stability is less than that of group I (Fig. 2). Nevertheless, conservation of the nucleotide sequences comprising the bottom part of the stem indicates that pairing of these bases may be important for recognition of group II ssi by n' protein. No significant homology was observed between the two groups except for the pentanucleotide CGCCG present within the conserved sequences of both groups, although the significance of this pentanucleotide is obscured by the fact that it is not present either in n'-pasB (ColEl) or in n'pas(F.f2a).
Three ssi Promote SS to RF Conversion by a G4 Type Mechanism and Are Directly Used by Primase-SS to RF replication in a fraction II extract on ssiE(F.fG), ssiB(RGK), and ssiA(R100) was independent of dnaB and dnaC functions ( Table I). The presence of extensive sequence homology among these three ssi predicted a common priming mechanism.3 Although substantial replication was observed in the @X174 reconstitution assay on these templates (Table II), none of the prepriming proteins were required (data not shown). Therefore, a reconstitution system for G4 DNA replication composed of SSB, primase, and DNA polymerase III holoenzyme was tested. The three chimeric phage DNAs were replicated efficiently in this system ( Table V). Omission of any one of the three components reduced the activity to a background level. These results indicate that primase directly recognizes these ssi (which is termed G site) and synthesizes RNA primers. ssiE(F.fG), ssiB(RGK), and ssiA(R1OO) will be renamed G site(F.f6), G site(RGK), and G site(RlOO), respectively.
RNA Primers Synthesized by Primase on G Sites-RNA a N. Nomura, H. Masai, and K. Arai, manuscript in preparation.
primers synthesized on the three G sites were directly analyzed. Primers of discrete length were detected on each of the three templates (Fig. 3). The lengths of the longest primers synthesized were 17 nucleotides for G site(F.f6) and G site(R6K) and 18 nucleotides for G site(R1OO). A longer primer RNA, nearly 30 nucleotides long, was occasionally detected on G site(R1OO) (Fig. 6, lane 3), although we have never seen primer RNAs of similar sizes on G site(F.f6) and G site(R6K).
Synthesis of RNA primers on G sites was dependent on the presence of SSB, as is the replication, indicating that productive recognition of G sites by primase requires coating of template DNA by SSB.
Location of 5' End Terminus of Primer RNAs-Priming by a G4 type mechanism on the G sites predicts that primer RNAs are synthesized at a specific site within the origin (4). The 5' ends of RNA primers synthesized by primase were localized by determining the nick site of the final RF11 products generated in vitro. On all the G site templates, the 5' ends of primer RNAs were mapped within or in the vicinity of the first conserved block sequences (Fig. 4). With G site(RlOO), the major RNA 5' end was mapped at the T residue at position +l in Fig. 5 (Fig. 4A, lane 5). This residue is the middle T of the CTG triplet, which is conserved also in the G4 origin and is where primer RNA is initiated in G4 replication (4,29). In G site(F.f6), the major 5' end was mapped at the C residue, 2 bases upstream of this T residue ( Fig. 4.4, lane I). In G site(RGK), the 5' ends of primer synthesized by purified primase were scattered in two regions separated by 5 bases (Fig. 4A, lane 3). The 5' end of the longest primer RNA was at the T residue of the conserved CTG triplet. Insertion of 4 bases immediately 5' to the first conserved block in the aligned G site sequences (Fig. 4B) may result in initiation at two different locations; however, only one cluster of 5' ends were observed with the products synthesized in fraction II (Fig. 4A, lane 4). There may be additional protein factor(s) that permit the precise recognition and initiation by primase on G site(R6K). In G site(F. f6) and G site(RlOO), 5' ends of RNA primers synthesized in a fraction II extract were mapped lo-12 bases downstream from those mapped with RF11 synthesized in the purified system (Fig. 4A, lanes 2 and 6), indicating that most of the RNA primers are removed in a fraction II, presumably by 5'+3' exonuclease activity of DNA polymerase I. Alkaline treatment of the products synthesized in the purified system prior to gel electrophoresis shifted the position of the 5' end by 12-14 bases (data not shown), indicating that the length of RNA primer when coupled to chain elongation is shorter than that synthesized in an uncoupled system (See Fig. 3) (30). Delineation of a G Site Required for Priming by Primase-In order to determine the maximum limit of G site required for priming in vitro, deletion derivatives of G site(R1OO) were constructed, and the activities of these templates in DNA replication and primer RNA synthesis were measured. Using restriction sites present in the insert DNA, two derivatives of G site(RlOO), RlOO-154 (from position +137 to -17) and RlOO-247 (+137 to -llO), were constructed. As the length of the insert became smaller, the plaque sizes decreased (data not shown). Parallel decrease of template activity in replication and primer RNA synthesis in vitro was observed (Table  VI; Fig. 6, lanes 1 and 2). However, both RlOO-247 and RlOO-154 directed synthesis of primer RNAs of the same length. Therefore, more systematic deletions were introduced from both ends of the insert DNA (Table VI). Deletion from the 3' end of the insert appears to decrease the replication activity according to the size of the deletion. When deletion reached position -44 (pHM7687), the replication activity was de-  creased to less than half of the control. An activity nearly 10% of the control still remained, even when deletion reached position -9 (pHM7685), only 8 bases away from the 5' end of the primer RNA. Replication activity was completely lost when deletion reached position +17 (pHM7716), passing the 5' end of the primer RNA. On the other hand, deletion from the 5' end of the insert abolished the replication activity when it reached position +99 (pHM7743), nearly 100 bases away from the 5' end of primer RNA, although deletion up to position +129 (pHM7738) had no effect on the activity. The deletion in pHM7743 removes a part of the consensus blocks, suggesting the importance of the conserved nucleotide sequences for recognition by primase. In keeping with this, the same deletion completely abolished primer RNA synthesis in uitro (Fig. 6, lane II), whereas primer RNA was still synthesized on pHM7685 containing only 8 bases upstream of the priming site, albeit at a reduced level (Fig. 6, lane 7). The sizes and pattern of primer RNAs were not altered in any of the deletions, except that the longest primer RNA was de-tected only with the parental G site(R1OO) (Fig. 6, lane 3). From these results, we concluded that the maximum limit of a G site for priming by primase is from position +128 to -8, which contains all the conserved sequences (Fig. 5). However, it is obvious that the sequences 3' to this region, although nonessential, increase efficiency of priming both in vivo and in vitro by as much as lo-fold. DISCUSSION Synthesis of primer RNA is a critical step in initiation of chain elongation. Biochemical studies of priming by using naturally occurring SS phages as model systems contributed to our understanding of enzymatic mechanisms of primer RNA synthesis (1,2). Subsequent studies indicated that priming mechanisms utilized by these phages actually function in priming of leading or lagging strand synthesis in replication of double-stranded DNA replicons. In replication of ColEl or its related plasmids, 4X174 type priming mediated by the primosome assembled at an n'-pas on the lagging strand RNA primers, synthesized on various G sites by primase in standard reaction mixtures either in the presence or absence of SSB, were analyzed by electrophoresis on a 15% denaturing polyacrylamide gel electrophoresis as described under "Materials and Methods." Template DNA in lanes 10 and 1 I is R199/G4 (34). Lanes 2, 5, and 8 are with twice the amount of primase. Less primer RNA synthesis on F. f6 is probably due to loss of some of the sample during the preparation, since the level of RNA synthesis as measured by using DEAE-cellulose filter paper was roughly the same among the three G sites (data not shown). template is responsible for efficient priming for the lagging strand synthesis (l&19).
On the other hand, G4 type priming appears to serve for priming of the leading strand synthesis at a unique site in replication of Rl plasmid (21). Both priming signals have been isolated as ssi by taking advantage of their ability to complement poor growth of a SS phage lacking the complementary strand origin (12)(13)(14)17). This fact lead us to assume that functionally important priming signals can be randomly isolated by this clear-plaque assay. Accordingly, six ssi from F, two from R6K, two from the vicinity of the basic replicon of RlOO, and one from downstream of the ColE2 origin were isolated. Biochemical characterization of priming on these ssi revealed that, with exception of two ssi, they either support 623174 type primosome assembly (n'-pas) or priming by primase directly (G site).
Two sequences, ssiD(F. f3) and ssiA(RGK), appear to adopt a novel priming mechanism in that they are not replicated in a reconstituted replication system for either type (66). Comparison of the nucleotide sequences of various n'-pas revealed the presence of two groups. Consensus hairpin structures, whose stems are mostly composed of conserved nucleotide sequences, could be drawn for each group. Furthermore, altered sequences in the stem are frequently compensated by changes in their pairing partners such that the pairing is maintained (Fig. 2), supporting the validity of the proposed stem and loop structure. Although different secondary structures can be drawn for group II n'-pas (31), the one shown in Fig. 2.4 better explains the results of mutational analysis (32, 33). All but one single-base mutations affecting the ATPase effector activity were mapped within the residues involved in in vitro. A, products synthesized in standard G4 SS to RF reconstitution assays (lanes I, 3, and 5) or in fraction II assays (lanes 2, 4, and 6) were purified, digested with N&I (lanes I, 2, 5, and 6) or with Sau3AI (lanes 3 and 4)  the base pairing in the stem. On the other hand, those singlebase mutations which have no effect on n'-pas activities were mapped on the unpaired residues except for one mutation (Fig. 2B). Furthermore, the second site revertants which reactivated the inert multiple-base mutant n'-pas restored the base pairing at the mutated positions in all the cases (Fig.  2, A and B). Alteration of C at position 15 to T also increases the number of pairings in the stem. These observations strongly support the idea that the stem-loop structures drawn in Fig. 2 are actually formed and that they are important for recognition by n' protein. It is more likely that n' protein recognizes a higher order structure of DNA involving hairpin structures rather than recognizing a single consensus sequence, as was previously predicted from mutational analysis of the n'-pas derived from pBR322 (32, 33). However, it is still possible that n' protein recognizes more than one specific sequences. It should also be noted that most of the residues directly in contact with n' protein, revealed by methylation protection experiments (31), fall into the conserved residues. Further analysis of interaction of n' protein with a variety of n'-pas may clarify the mode by which it recognizes its target sequences. Extensive sequence homology was discovered among three G4 type ssi (G sites)." Deletion analysis indicated that these conserved sequences are important for functional recognition by primase. Conservation of the priming site also indicates that primase recognizes a common structural feature shared by these three G sites. A 128-base region downstream of the priming site is essential for priming, whereas only 8 bases upstream of the priming are sufficient, a situation similar to the G4 phage origin (34), although the presence of the sequences further upstream apparently increases the efficiency of priming both in vivo and in vitro. In spite of the identity of the priming mechanism, sequence homology is minimal between the three plasmid-derived G sites and the complementary strand origin of G4 phage. Stem and loop structures found near the G4 origin are also not detected within the newly isolated G sites. The trinucleotide CTG, which is conserved between the three G sites and the G4 origin and the middle T of which is the 5' end of primer RNA, is frequently found 10 to 11 bases upstream from the RNA/DNA junctions in nascent OKAZAKI fragments from near oriC (35) and oriX (36). The presence of CTG may be important for primer RNA synthesis by primase at a replication fork. Recently, mutational analysis of the G4 origin established that CTG is critically important for priming in vivo (37).
We believe that both n'-pas and G sites play functional roles in replication of double-stranded DNA replicons from which they are derived. The frequent presence of ssi near replication origins is also indicative of their role in priming. We can now speculate as to what the roles of some ssi are, based on their priming modes and locations together with known replication strategies taken by individual replicons.
Based on the analysis of modes of priming on duplex DNA, we propose four different modes of initiation, depending on the ways in which the primosome is assembled.
(i) oriC type (bidirectional): the absence of efficient ssi near oriC4 suggests that the primosome assembly at oriC is sufficient for priming of both leading and lagging strand syntheses. The loading of dnaB protein onto each strand of the template DNA at the origin through an "open complex" (38-40) and subsequent unwinding in both directions (41) ensure priming and replication in a bidirectional fashion. Therefore, any additional ssi for leading and lagging strand syntheses need not be present. This mode of bidirectional DNA replication by two dnaB molecules, each located on each strand, was originally proposed by McMacken et al. (42).
(ii) Rl type (unidirectional): in unidirectionally replicating replicons such as Rl or RlOO, the primosome assembly at the origins permits the loading of only one dnaB protein, which supports unidirectional expansion of a replication fork together with priming of lagging strand. The leading strand initiation is provided by a G site present downstream of the origin (20, 21). Therefore, the G site(R100) represents the priming signal for the leading strand synthesis. Unidirectional replication from orb of R6K plasmid (43) is likely to involve a similar priming on a G site. In this case, a replication fork moving unidirectionally must be established by loading one dnaB protein onto DNA by some mechanism. This may be achieved by transferring dnaB protein from oriy (44). As the replication fork traverses, the priming signal is activated, and primase can make an RNA primer for the leading strand synthesis at the G site(R6K) (Fig. 7). The direction of replication from orb (43) is consistent with that of priming at the G site(R6K).
(iii) Two-step bidirectional type: two @X174 type ssi, n'pas(F.f5) and n'-pas(F.t7), are present 60-70 bp to the left of AT-rich sequences or GATC clusters present within the minimal origin in such an orientation that the primosome assembled at these n'-pas moves toward the origin (45-47). We presume that the activation of the origins leads to the loading of one dnaB protein in much the same way as at origins of the Rl type and that it migrates unidirectionally to the left on the lagging strand template, while occasionally synthesizing primer RNAs (Fig. 8). The n'-pas on the leading strand template is activated when it is transiently exposed as SS DNA as the replication fork passes through it. The primosome, assembled at the n'-pas, now moves along the leading strand template toward the origin, eventually passes through it, and creates a right-bound replication fork, establishing bidirectional replication.
The primosome not only provides priming for the lagging strand synthesis on the bottom strand, but it can also initiate left-bound leading strand synthesis. The previous report that replication from OFi on the f5 fragment is unidirectional (48) may indicate that the left to right fork unwinding by dnaB helicase is blocked by a Rep protein complexed at the origin, as the terbinding protein bound to the ter sequence blocks helicase action of dnaB protein (49, 50). The direction of replication from RepFIB on the f7 fragment is not known. A similar twostep bidirectional replication model can be applied to oriy of R6K, since ssiA(RGK), located at the identical position relative to the origin as in Fig. 8, can support assembly of a similar mobile "primosome" with a different set of prepriming proteins (66).
(iv) ColEl type (unidirectional): in this class, the leading strand synthesis is first initiated at a specific site without involving loading of dnaB protein ( Fig. 9) (51, 52). The structure of the origins is consistent with this notion in that they lack any AT-rich sequences or GATC clusters through which dnaB can be guided onto DNA. An essential role of DNA polymerase I (53) is in keeping with the necessity to unwind duplex DNA by its strand displacement activity in and n'-pas(F*f7). Marks are the same as in Fig. 7 indicates the replication origin and the thick arrow represents a n'pas with the 5' to 3' direction.
A, RNA polymerase or the plasmidencoded Rep protein recognizes the origin and makes a primer RNA, which is elongated by DNA polymerase I for the leading strand synthesis (51,52). B, the primosome is assembled at the n/-pas, which is exposed as SS DNA as a result of the leading strand synthesis. C, the primosome or its subassembly migrates on the lagging strand template and propagates the replication fork toward the right, while synthesizing RNA primers for OKAZAKI fragments.
the absence of dnaB protein. A mobile primosome, assembled at the n'-pasA(ColE1) or n'-pas(ColEB), migrates on the lagging strand template in the direction of fork unwinding and establishes an efficient replication fork, which can synthesize primer RNAs for OKAZAKI fragments (18,19). The role of n'-pas in lagging strand synthesis of ColEl-type plasmid replication had been previously proposed (12, 54). Although the ssi described above are likely to play impor-tant roles in priming DNA replication, it should also be noted that they are usually located outside the minimum sequences required for autonomous replication and that some alternative, less efficient mode of priming must be operating in their absence.
The term primosome originally referred to the protein complex assembled at the n'-pas for 4X174 type priming (55). However, in view of the presence of similar primosomes requiring different prepriming proteins (56, 66) and prepriming complexes which can be isolated at the duplex origins (57, 58), it would be more appropriate to redefine the primosome as a protein complex in general which is capable of priming and duplex unwinding at the replication fork. We think that a primosome assembled at a duplex origin containing a dnaB loading site and that assembled at a SS primosome assembly site such as n'-pas are equivalent in their ability to establish a functional replication fork capable of fork unwinding and priming of lagging strand. Primosomes traveling bidirectionally are generated at the duplex origins in some cases (oriC or X) (41, 59), while only one primosome traveling unidirectionally is generated at others (Rl and probably pSC101, Fori2, and RGKorir, etc.). At present, it is not known what determines the mode of dnaB loading; i.e. the number of dnaB/ dnaC protein complexes guided onto a template DNA. In contrast to priming by the primosome, priming on G sites appears to serve soley for the leading strand initiation, which requires only a single priming event.
Roles of other ssi are not clear at this moment. n'-pas(F. f2a) and n '-pas(F.f2b) are located next to each other on the opposite strands. If the primosomes could be somehow assembled at each ssi on both strands, it would create the replication forks moving in both directions. This region could represent a silent replication origin, which is activated under some particular condition. G site(F.f6), which is located close to oriT (60), a nick site for rolling circle type transfer replication, may function in transfer DNA replication.
However, its precise function is not clear. n  lies outside the basic replicon required for regulated autonomous replication of RlOO, which makes it unlikely that it plays an essential role in vegetative replication. The mechanism of priming on ssiD(F. f3) is not clear, nor is its function. Further analysis of replication of individual replicons will clarify the function of these ssi.