37
38
described in section 2.9, DNA synthesis is continuous on the leading-strand but discontinuous (Okazaki fragments) on the lagging-strand. The synthesis of the two strands is coupled as in T7, but replication may proceed either bi- or more commonly unidirectionally (Figure 2.9). The coupled synthesis of leading and lagging-strands on a circular molecule results in replication intermediates, which look like the greek theta (Θ).
Figure 2.9. Uni- (A) and bidirectional (B) replication. Bidirectional replication requires always two independently recruited replisomes.
Replication terminates at defined sequences, and the terminus is actively determined by protein interactions. One of the best characterized termini are ter sequences in plasmid R6K, which function as a terminus for unidirectional theta replication. The ter sequence is a binding site for the termination protein Tus and interferes with the helicase progression in an orientation-dependent manner. The replication is sequential; strand-synchronous synthesis first proceeds to the terminus in just one direction, terminates and subsequently the origin initiates to the opposite direction and progresses to completion (Figure 2.10. [Lovett et al. 1975]).
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Figure 2.10. Sequential unidirectional theta replication of a bacterial plasmid.
Sequences called terH, which arrest lagging-strand synthesis, are found in the plasmid ColE1, where they induce termination through a stable transcript hybridized at the site. The stalling of the replication fork may result from the presence of an unhybridized run of RNA (Solar et al.
1998).
Catenated molecules containing gaps in the daughter-strands are usually the last step of replication. The catenanes can be resolved by either type I or type II topoisomerases; however it is likely that a class II topoisomerase – TopoIV – has a specialized role for unlinking the daughter replicons in vivo. Maturation of the open-circular gapped forms into supercoiled molecules is further facilitated by DNA polymerase for gap filling and DNA gyrase for introduction of supercoils. With e.g. the plasmid R1 it seems that the maturation of newly replicated DNA is a slow process, preventing the direct utilization of the newly replicated molecules.
In theta replicating plasmids, homologous recombination between daughter molecules can also occur. Replication intermediates provide ideal substrates for recombination and these events may result in the generation of dimeric molecules (Nordström 1983).
40 2.12.2 Strand displacement replication
Strand displacement replication (SDR) is rare and the best studied examples of such plasmids are the members of the promiscuous IncQ family. These plasmids encode three proteins required for initiation of DNA replication: RepA, RepB and RepC (Scherzinger et al. 1991, Solar et al. 1998).
The minimal ori includes three identical sequence repeats plus other typical palindromic sites and inverted repeats. The identical repeats act as RepC binding sites from which RepC together with RepA helicase can induce partial opening of the sequence, causing the exposed inverted repeats to form hairpin structures, which can be recognized by the plasmid encoded RepB primase. The replication initiation is therefore independent of any host initiation factors, requiring only host DNA Pol III and SSB for replication.
Initiation at either of the inverted repeats can occur independently and result in continuous replication with the RepA helicase facilitating the displacement of the parental strand.
Replication initiating from both inverted repeats would result in a theta-shaped intermediate in the overlapping regions and displacement loops beyond these regions (Figure 2.11). The end products of SDR are completely or partially single-stranded displaced circles and double-stranded supercoils.
Replication of the displaced strand can also be initiated from the exposed hairpin structure of the inverted repeat.
Figure 2.11. Strand displacement replication from either one (A) or two (B) origins on the different strands.
41 2.12.3 Rolling circle replication (RCR)
Replication by RCR is unidirectional and strand-asymmetric, the leading and lagging-strand synthesis being uncoupled. Most of the RCR plasmids are smaller than 10 kb and can exist as either ssDNA or dsDNA circles when mature (Solar et al. 1998).
RCR is initiated by a plasmid encoded Rep protein, which introduces a site specific nick on the plus-strand of the plasmid in the region termed double-strand origin dso. The dso has two loci, bind and nic, of which the former is involved in the sequence specific binding of the Rep protein and the latter represents a conserved sequence that is nicked by the protein. The two loci can be adjacent or separated by up to 100 bp. The nicking at nic leaves a free 3´-OH that can prime replication by host replication proteins. As a result, the elongation of the end displaces the parental plus-strand and continues until the dso is met again. Thus the intermediate products of RCR are a dsDNA molecule constituting parental minus-strand and newly synthesized plus-strand and a single-stranded displaced parental plus-strand (Figure 2.12).
Figure 2.12. Rolling circle replication. The circular template duplex is nicked and the DNA- polymerase initiates synthesis from the free 3´–end of the nick. DNA synthesis replaces the nascent strand from the template. Several rounds of replication can occur producing concatemeric replication products. The joining and the subsequent ligation of the displaced strand is catalyzed by the Rep protein.
Besides initiating RCR, Rep protein is also involved in replication termination.
Replication of the leading-strand proceeds more than a full round beyond the nick site, enabling hairpin formation by an inverted repeat region at dso. Rep cleaves the hairpin and facilitates the
42
ligation reaction of the newly synthesized 3´-OH with the 5´ of the nick and liberating the circular dsDNA product. Continuous replication of the leading-strand is possible, producing head-to-tail concatemers of multiple genome sizes that are later processed into circular monomers by recombination.
The final stage of RCR is the replication of the displaced single-stranded parental (+) strand into dsDNA. The replication is primed by RNA (pRNA) and in most cases evidence suggests that host RNA polymerase (RNAP) is involved. RNAP initiates transcription from the plasmid single-strand origin sso sequence, resulting in a 20 nt RNA primer, which is in turn elongated by the host replication machinery. The newly synthesized dsDNA plasmids are subsequently supercoiled by DNA gyrase into their active form.