in Vitro of Three Replication Origins of the Antibiotic Resistance Plasmid

Replicating molecules of plasmid RSF1040, a deletion mutant of R6K, were synthesized in vitro and analyzed by electron microscopy. Initiation of replication occurs at three unique sites, ori alpha, ori beta, and ori gamma, within a 3900-base pair segment of the R6K genome. These sites are indistinguishable from the origins that are active in vivo. Frequencies of initiation at these three origins, however, are different from those observed in vivo. Replication proceeds unidirectionally in either direction from ori beta and ori gamma and in one direction from ori alpha. The replication terminus of the R6K genome is inactive in the in vitro system.

Replicating molecules of plasmid RSF1040, a deletion mutant of R6K, were synthesized in vitro and analyzed by electron microscopy. Initiation of replication occurs at three unique sites, oria, 0x-i/3, and oriy, within a 3900base pair segment of the R6K genome. These sites are indistinguishable from the origins that are active in ho.
Frequencies of initiation at these three origins, however, are different from those observed in ho. Replication proceeds unidirectionally in either direction from ori/3 and oriy and in one direction from oria. The replication terminus of the R6K genome is inactive in the in vitro system.
. Initiation of prokaryotic DNA replication usually occurs at a unique site on the DNA molecule. However, several prokaryotic replicons have been reported to have more than one origin of replication (l-3). In these cases, one of the replication origins is used predominantly with the other origin sites used infrequently or only when the normal origin site is deleted or when the structure of the replicon is otherwise modified. It is highly interesting to determine the genetic and biochemical nature of the control of these multiple origins and their usage in the initiation step of replication. Plasmid R6K is a conjugative plasmid of molecular weight 38 kilohase pairs and codes for resistance to streptomycin and ampicillin (4). This plasmid and its deletion derivative, RSF1040, have been shown to possess two origins of replication in viva, which are separated by approximately 3900 base pairs (5,6). Replication from either one of the two origins, designated (Y and /3, is unusual in that it proceeds unidirectionally towards an asymmetric terminus, followed by replication from the same origin in the opposite direction (5,7). An in vitro system capable of replicating R6K DNA has been developed (8,9). It was established with this system that an RGK-encoded protein, r protein, is required for the initiation of R6K DNA replication.
Preliminary results suggest that this protein associates with the template DNA as part of the initiation event (9). Studies with this in vitro system are providing a very useful approach to understanding the mech- level of replicative intermediates in the DNA preparation, the following synchronization process for the initiation of replication in vitro was carried out. The standard reaction mixture lacking all dNTPs was preincubated for 10 or 15 min to form initiation complexes involving Q protein and to facilitate nascent RNA synthesis that is necessary for the initiation of R6K replication. The four dNTPs including [3H]TTP were then added to initiate DNA synthesis. The reaction was terminated after 1.5 min, and the labeled products were centrifuged to equilibrium in an ethidium bromide/CsCl density gradient. A majority of the [3H]DNA banded at a position between supercoiled and open circular DNA (Fig. la). To further purify the replicating molecules, the intermediate density fractions were pooled and recentrifuged. The replicating molecules once again banded at the position of intermediate density (Fig. 1b). Under these pulse-label conditions, 14% of the pulsed radioactivity in RSF1040 DNA was recovered in the replicative intermediate fraction.
Analysis of Eco RI-cleaved Replicative Intermediates-Purified pulse-labeled RSF1040 DNA in the intermediate density position was treated with Eco RI which cleaves RSF1040 DNA at a single unique site (14) and observed by electron microscopy. The replicating molecules are schematically represented in the order of increasing extent of replication on the basis of the known positions of the two origins observed in vivo and in correspondence with a third origin of replication (Fig. 2). R6K DNA also was used as a template in this in vitro system. Unlike RSF1040, R6K DNA contains two Eco RI sites that are asymmetrically located. The origins of replication of this plasmid in vivo are located in the smaller (B) fragment generated by Eco RI cleavage (5, 14). In the in vitro system, all origins of replication again were located by electron microscopy on the B fragment and in no case was a replication origin detected on the larger (A) Eco RI fragment. It has been shown that the in vivo replication origins of RSF1040 correspond to those of R6K (14). Therefore, it is most likely that the replication origins of RSF1040 in vitro are located on the same side near the Eco RI site as shown in Fig. 2.
In order to determine the precise location of the origin sites in Fig. 2, a histogram of the frequency of initiation sites on the genome was made (Fig. 3). It is clear that three replication origins, designated a, p, and y, are active in RSF1040 DNA. The a, y, and p origins are located at 25.2% 2 1.3%, 33.9% f 1.4%, and 39.4% k 0.9%, respectively, from the Eco RI site of RSF1040 DNA. The three origins are clustered in a region equivalent to 14.2% of the RSF1040 genome and 9.0% of the R6K genome. Of the 121 replicating molecules, only 1 molecule was found to use both oria and oriy, simultaneously.
As shown in Fig. 4, the sites of the initiation of replication in vitro have been positioned on the R6K and RSF1040 restriction maps. The a, y, and /? origins are located on the HindIII fragment 4, around the junction of fragments 9 and 4, and around the junction of fragments 2 and 15, respectively. The in vivo results on the replication of R6K and RSF1040 derivatives has shown that oria is located midway on HindIII fragment 4, while or$ is located at the end of fragment 2 that Linear replicating molecules were arranged in order of increasing extent of replication from three origins. ter, specific terminus which is active in vivo (5, 7). is adjacent to fragment 15 (6). Therefore, two of the three origins that have been observed in the in vitro system compare well with the positions of oria and or$ determined by the in vivo studies. The y origin of replication on RSF1040 that appears to be used frequently in vitro recently has also been observed during in vivo replication (10).

Three Replication Origins in Vitro on
Frequency of Origin Usage and Directionality-The analysis of RSF1040 and R6K DNA replicated in vivo has indicated that initiation occurred at oria more frequently than at orip (6). In the case of the initiation in vitro, the frequencies of initiation from these two origins is quite different (Table I). Initiation occurs at the frequencies of 24%, 43%, and 37% from the orict, orip, and oriy, respectively. Replication initiated from oria was observed to proceed only counterclockwise for the molecules examined. Replication from or$ or oriy, however, proceeds in either direction, but only in one direction at least initially. It appears that replication proceeds counterclockwise from oria, orip, or oriy at almost the same frequency among the molecules examined. Bidirectional replication from any of the three origins was not observed.   11 9 37

Inactivity of the Specific Terminus for R6K Replication in
Based on the analysis of 121 replicative intermediate molecules.
or orip (5, 7 ) . The specific terminus appears to be inactive in vitro under the conditions employed since, as shown in Fig. 2, for 34 of the total 121 replicating molecules, the replication fork has passed the terminus site located at 55% from the Eco RI cleavage site.

DISCUSSION
It has been found that plasmid RSF1040 possesses three origins of replication that are functional in replication in vitro. The locations of the three origins are within a 3900-base pair segment of the plasmid genome. Two of the observed origins appear to be identical in position with origins ct and fl previously shown to be functional in replication in vivo (6). The third origin, y, recently also has been shown to be active in vivo (10). The y origin appears to be identical with the 360base pair origin segment isolated from R6K DNA and functional in E. coli cells when the n protein is provided in trans (16). The y origin is located in the region of the junction of Hind111 fragments 9 and 4. Initiation of replication at the three origins in vitro required an extract prepared from R6Kcontaining cells. While initiation of DNA replication at the y origin required the n protein in vivo (16) and in vitro,' there is no direct evidence as yet whether or not this protein and/or other R6K-encoded proteins are required for initiation of the a and p origins in vivo or in vitro. However, the fact that the ct origin requires HindIII fragments 15 and 9 in cis for activity suggests that the n protein is required for origin activity (6). HindIII fragments 9 and 15 encode n protein (16).
The reason for the differences in frequency of origin usage and directionality found in the in vitro studies presented here (Table I) and in the in vivo analyses (6, 10) is unclear. It is possible that selectivity of the origins for initiation might be lost due to the preincubation step carried out to synchronize the initiation event. It was found that initiation occurred at oria more frequently after preincubation for 10 min rather than 15 min. It is also possible that selection of an origin site is influenced by a specific secondary or tertiary structure of this region of R6K that is generated by interaction with other cellular components in intact cells. Replication of RSF1040 in vitro proceeded unidirectionally from all three origins (Fig. 2). The in vivo results indicate that both oria and orip have a potential to initiate replication sequentially in both directions (5,6). In the case of oria, only counterclockwise replication was observed in vitro. A similar pattern of replication is observed for oria in vivo when the terminus is deleted (6). For or$ replication proceeded in both directions at almost the same frequency, while the major initial direction for this origin of replication in vivo is clockwise. Another example of unidirectional replication in both directions comes from the analysis of replicative intermediates of mini-ColEl, (pVH51) in vivo (17), and RSFlOlO (18). In these cases, bidirectional replication also was observed.
In the in vitro system employed, the terminus of RSF1040

Three Replication Origins in Vitro on RSF1040
did not function, although previous in vitro results had suggested an active terminus (8). Termination of replication may require additional components, for example a membrane site and/or other proteins that are deficient in this system, in addition to the specific nucleotide sequence. The requirements for an active termination will be examined using this in vitro replication system.