Multiple Mechanisms Exist to Prevent Premature Reinitiation of Replication

KEY CONCEPTS
- SeqA binds to hemimethylated DNA and is required for delaying rereplication.
- SeqA can interact with DnaA.
- As the origins are hemimethylated, they bind to the cell membrane and might be unavailable to methylases.
- The dat locus contains DnaA-binding sites that titrate availability of DnaA protein.
- Hda protein is recruited to the replication origin to convert DnaA-ATP to DnaA-ADP.
Replication in bacteria and in eukaryotes is licensed and permitted to occur only once per cell cycle. Each replicon is allowed to fire only once. What mechanisms are in place to ensure reinitiation does not occur? Because it is critical to maintain genomic integrity, multiple mechanisms exist to ensure that each replicon fires once, and only once, during each cell cycle.
As described in the section Methylation of the Bacterial Origin Regulates Initiation earlier in this chapter, the E. coli oriC is fully methylated at the beginning of replication. After semiconservative replication has occurred, oriC is hemimethylated and remains in that condition for approximately 13 minutes. What is responsible for this delay in remethylation at oriC? 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 can initiate binding when the DNA becomes hemimethylated, at which point 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. This would explain the genetic interactions between seqA and dnaA.
As the only member of the replication apparatus uniquely required at the origin, DnaA has attracted much attention. DnaA is a target for several regulatory systems. It might be that no one of these systems alone is adequate to control frequency of initiation, but that when combined they achieve the desired result. Some mutations in dnaA render replication asynchronous, which suggests that DnaA could be the “titrator” or “clock” that measures the number of origins relative to cell mass. Overproduction of DnaA yields conflicting results, which vary from no effect to causing initiation to take place at reduced mass.

The availability of the amount of DnaA for binding at the origin is the result of competition for its binding to other sites on the chromosome. In particular, a locus called dat has a large concentration of DnaA-binding sites. It binds a larger number of DnaA molecules than the origin. Deletion of dat causes initiation to occur more frequently. This significantly increases the amount of DnaA available to the origin, but researchers do not yet understand exactly what role this might play in controlling the timing of initiation. It has been difficult to identify the protein component(s) that mediate membrane attachment of oriC. 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.
Researchers 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 membraneassociated inhibitor, but when the origin becomes fully methylated, the inhibitor is displaced by DnaA associated with the membrane. Because DnaA is the initiator that triggers a replication cycle, the key event will be its accumulation at the origin to a critical level. There are no cyclic variations in the overall concentration or expression of DnaA, which suggests that local events must be responsible. To be active in initiating replication, DnaA must be in the ATP-bound form. Thus, hydrolysis of ATP to ADP by DnaA has the potential to regulate its own activity. Although DnaA has a weak intrinsic ATPase activity that converts the ATP to ADP, this is enhanced by a factor termed Hda. In a conceptually elegant feedback loop, Hda is recruited to a replication origin via the βsubunit of the DNA polymerase. Thus, only when the origin has been activated and the full replication machinery assembled is Hda recruited, it acts to switch off DnaA, preventing a second round of replication.
The full scope of the system used to control reinitiation is not clear, but multiple mechanisms are involved: physical sequestration of the origin, delay in remethylation, competition for DnaA binding, hydrolysis of DnaA-bound ATP, and 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. Indeed, we still have to come to grips with the central issue of which feature has the basic responsibility for timing. 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. One aspect of the control might lie in the observation that hemimethylation of oriC is required for its association with cell membranes in vitro. This might reflect a physical repositioning to a region of the cell that is not permissive for replication initiation.