Genes VII

What feature of a bacterial (or plasmid) origin ensures that it is used to initiate replication only once per cycle? Is initiation associated with some change that marks the origin so that a replicated origin can be distinguished from a nonreplicated origin?

Some sequences that are used for this purpose are included in the origin. oriC contains 11 copies of the sequence ^GATC_CTAG, which is a target for methylation at the N⁶ position of adenine by the Dam methylase. The reaction is illustrated in Figure 13.30.

Before replication, the palindromic target site is methylated on the adenines of each strand. Replication inserts the normal (nonmodified) bases into the daughter strands, generating hemimethylated DNA, in which one strand is methylated and one strand is unmethylated. So the replication event converts dam target sites from fully methylated to hemimethylated condition.

What is the consequence for replication? The ability of a plasmid relying upon oriC to replicate in dam ^V E. coli depends on its state of methylation. If the plasmid is methylated, it undergoes a single round of replication, and then the hemimethylated products accumulate, as described in Figure 13.31. So a hemimethylated origin cannot be used to initiate a replication cycle.

Two explanations suggest themselves. Initiation may require full methylation of the Dam target sites in the origin. Or initiation may be inhibited by hemimethylation of these sites. The latter seems to be the case, because an origin of nonmethylated DNA can function effectively.

So hemimethylated origins cannot initiate again until the Dam methylase has converted them into fully methylated origins. The GATC sites at the origin remain hemimethylated for ~13 minutes after replication. This long period is unusual, because at typical GATC sites elsewhere in the genome, remethylation begins immediately (<1.5 min) following replication. One other region behaves like oriC; the promoter of the dnaA gene also shows a delay before remethylation begins.

While it is hemimethylated, the dnaA promoter is repressed, which causes a reduction in the level of DnaA protein. So the origin itself is inert, and production of the crucial initiator protein is repressed, during this period.

What is responsible for the delay in remethylation at oriC and dnaA? The most likely explanation is that these regions are sequestered in a form in which they are inaccessible to the dam methylase.

A circuit responsible for controlling reuse of origins is identified by mutations in the gene seqA. The mutants reduce the delay in remethylation at both oriC and dnaA. As a result, they initiate DNA replication too soon, thereby accumulating an excessive number of origins. This suggests that seqA is part of a negative regulatory circuit that prevents origins from being remethylated. SeqA binds to hemimethylated DNA more strongly than to fully methylated DNA. It may initiate binding when the DNA becomes hemimethylated, and then its continued presence prevents formation of an open complex at the origin. SeqA does not have specificity for the oriC sequence, and it seems likely that this is conferred by DnaA protein, which would explain genetic interactions between seqA and dnaA.

Hemimethylation of the GATC sequences in the origin is required for its association with the cell membrane in vitro. Hemimethylated oriC DNA binds to the membranes, but DNA that is fully methylated does not bind. One possibility is that membrane association is involved in controlling the activity of the origin. This function could be separate from any role that the membrane plays in segregation (see Figure 12.26). Association with the membrane could prevent reinitiation from occurring prematurely, either indirectly because the origins are sequestered or directly because some component at the membrane inhibits the reaction. The properties of the membrane fraction suggest that it includes components that regulate replication. An inhibitor is found in this fraction that competes with DnaA protein. This inhibitor can prevent initiation of replication only if it is added to an in vitro system before DnaA protein. This suggests the model of Figure 13.32, in which the inhibitor specifically recognizes hemimethylated DNA and prevents DnaA from binding. When the DNA is remethylated, the inhibitor is released, and DnaA now is free to initiate replication. If the inhibitor is associated with the membrane, then association and dissociation of DNA with the membrane may be involved in the control of replication.

The full scope of the system used to control reinitiation is not clear, but several mechanisms may be involved: physical sequestration of the origin; delay in remethylation; inhibition of DnaA binding; repression of dnaA transcription. It is not immediately obvious which of these events cause the others, and whether their effects on initiation are direct or indirect.

We still have to come to grips with the central issue of which feature has the basic responsibility for timing. One possibility is that attachment to the membrane occurs at initiation, and that assembly of some large structure is required to release the DNA. The period of sequestration appears to increase with the length of the cell cycle, which suggests that it directly reflects the clock that controls reinitiation.

It has been extremely difficult to identify the protein component(s) that mediate membrane-attachment. A hint that this is a function of DnaA is provided by its response to phospholipids. Phospholipids promote the exchange of ATP with ADP bound to DnaA. We do not know what role this plays in controlling the activity of DnaA (which requires ATP), but the reaction implies that DnaA is likely to interact with the membrane. This would imply that more than one event is involved in associating with the membrane. Perhaps a hemimethylated origin is bound by the membrane-associated inhibitor, but when the origin becomes fully methylated, the inhibitor is displaced by DnaA associated with the membrane.