Involvement of Chaperones in the Control of DNA Replication of Bacterial Plasmids

Das N and Chattoraj DK. Origin pairing (“handcuffing”) and unpairing in the control of P1 plasmid replication. Mol Microbiol 54: 836–49, 2004.

hromosomal DNA replication is a highly controlled process in all growing cells. This is also true for multicopy plasmids of bacteria. If the plasmid copy number is not controlled, plasmid-free cells could result. The risk of generating plasmid-free cells increases with reductions in the copy number, but too high a copy number is also undesirable. Cells with too high a copy number grow more slowly, as plasmids are a metabolic burden to the host. These slow-growing cells can be outnumbered by faster-growing plasmid-free cells. Therefore, for stable maintenance, it is essential that plasmids maintain their copy number within narrow limits. They achieve this with remarkable economy using one or two plasmid-encoded genes.

Extrachromosomal existence, although risky, allows plasmids the opportunity for horizontal transfer to other species and genera. A classic example is the movement of the Ti plasmid from Agrobacterium to the nucleus of plant cells where they cause crown gall tumor. It is therefore not surprising that the host factors upon which plasmids depend are often well-conserved and ubiquitous proteins, such as molecular chaperones.

The involvement of molecular chaperones in DNA replication initiation has been known for quite some time from studies on bacteriophages and plasmids. In the best-studied cases, the chaperones remodel the initiators, resulting in activation of the initiator itself (Wickner S et al. Nature 350: 165–7, 1991) or release of replication factors trapped in an inactive state (Alfano C and McMacken R. J Biol Chem 264: 10699–708, 1989).

Two mechanisms are generally found to control the replication of bacterial plasmids. The first one is by antisense RNA. It was at the NIH that antisense control was discovered by Tomizawa J. and Itoh T. in studies of ColE1 (Proc Natl Acad Sci U S A 78: 6096–100, 1981). The second one, which we have studied in plasmid P1, is achieved by DNA repeats (iterons; block arrows, Figure 1) that bind the only plasmid-encoded initiator protein, Rep. The binding of Rep to iterons not only allows replication initiation but is also involved in controlling the frequency of replication initiation in the cell cycle. The control involves increasing the replication frequency in cells with too few plasmids and decreasing it in cells with too many plasmids (Das N et al. Proc Natl Acad Sci U S A 102: 2856–61, 2005). It appears that this inverse relationship of replication frequency to copy number (negative feedback control), which is central to maintaining the copy number within narrow limits, can be achieved by simple Rep dimerization and its dissociation with the help of chaperones.

Figure 1. Control of initiation of DNA replication by chaperone-mediated dissociation of initiator dimers. Initiator (Rep) binds to its multiple sites (iterons; block arrows) in the origin as a monomer. Saturation of the binding sites leads to origin folding that absorbs one superhelical turn of DNA and opens the adjacent AT-rich region, which encourages initiation. Upon replication, the initiator monomers are titrated by the daughter origins so that no origin is saturated. Replication also causes an increase of Rep synthesis. The nascent Rep dimerizes readily and competes with monomers for origin binding and/or mediates origin pairing. These events prevent origin folding. Chaperone-mediated dimer dissociation and increase of monomer allow origin saturation and folding, which preclude origin pairing. The replication cycle can thus repeat.

Rep is active as an initiator only as a monomer. However, Rep dimers readily form and require remodeling by chaperones (DnaK, DnaJ, and GrpE) to dissociate and serve as initiators. Our study shows that dimerization not only reduces monomers but could play an active role in control as well.

We believe a primary mechanism for negative control is by pairing of Rep-bound daughter origins (handcuffing). Pairing apparently prevents origin folding, which opens the origin, a prerequisite for replication initiation. Pairing of DNA sites is common in transcriptional repression and activation both in bacteria and eukaryotes. Pairing must be reversed if replication is to resume. In transcriptional control, pairing is reversed simply by adding inducer or depleting the pairing protein. In our case, the chaperones seem to do the trick.

When plasmid copy number increases, the increase in rep genes also raises total Rep concentration and thereby encourages more dimer formation. In fact, the dimer-to-monomer equilibrium ratio increases with increases in total protein. The dimers serve as inhibitors by effecting handcuffing and/or by directly binding to iterons. By converting dimers to monomers, the chaperones help by both reducing handcuffing and aiding origin saturation by increasing monomer concentration.

Replication control must ensure initiation but prevent premature reinitiation. Chaperones, by modulating the dimer-to-monomer (inhibitor-to-initiator) ratio, achieve both these goals. In this scheme, the economy of control does not seem to compromise the efficiency of control.

Dhruba K. Chattoraj, PhD
Principal Investigator
Laboratory of Biochemistry
NCI-Bethesda, Bldg. 37/Rm. 6044
Tel: 301-496-9194
Fax: 301-480-1493
chattoraj@nih.gov