# 60 Years of Studies into the Initiation of Chromosome Replication in Bacteria
## 3 The DnaA Protein
### Mechanisms Regulating Initiation
#### 3.4.6 SeqA  
The control over initiation could be based on the quality or the quantity of DnaA itself (see below, Section 5.2 \"The DnaA-ATP:DnaA-ADP Conundrum\"). Another mechanism for this control could be based on the accessibility of _oriC_ to DnaA. The involvement of the membrane in this mechanism was found by [PERSON]'s group [73], which showed that _oriC_ only bound to the membrane when _oriC_ was hemimethylated at Dam sites (GA-N5 methyl-TC), which are separated by 10, 21, or 32 bp (corresponding to one, two, or three helical turns of the DNA), and which are more frequent in _oriC_ than elsewhere on the chromosome [74]. [PERSON] et al. then showed that this membrane preparation inhibits in vitro replication of _oriC_ plasmids only when _oriC_ is in the hemimethylated state [75]. Subsequently, [PERSON]'s group and, independently, [PERSON]'s group [76] isolated the protein responsible for this interaction, and the former group identified the gene, which they termed _seqA_[77]. They demonstrated that SeqA binds preferentially to hemimethylated Dam sites rather than to fully methylated or unmethylated Dam sites. SeqA dissociation from _oriC_ is due to the complete Dam-methylation of the latter [78; 79]. Another important SeqA function is related to the replication hyperstructure, where SeqA tracks behind the forks [80] and perhaps brings together genes associated with DNA replication and repair [81], which helps organize the cohesion of daughter chromosomes [82], again using its affinity for hemimethylated GATC sites.  
The inhibition of the replication of hemimethylated _oriC_ plasmids by the SeqA protein has been shown by [PERSON]'s group to be due to its occupation of the DnaA boxes containing Dam sites (R5M, I2, and I3), which prevents DnaA from binding to these boxes. Indeed, in _dnaC_ cells, where the initiation of replication is suspended at an early stage, they found that DnaA bound to the high-affinity R1, R2, and R4 boxes but not to the lower-affinity R5M, I2, and I3 sites. The absence of DnaA binding to the R5M box and I2 and I3 sites may also be related to their location close to the IHF- and Fis-binding sites, so it is not surprising that mutants affected in these binding sites have asynchronous initiations [79].  
In vivo, _oriC_ sequestration via its Dam sites lasts for around 1/3 of the cell cycle [83], after which SeqA detaches from the region. Since this sequestration relies on the lower-affinity DnaA boxes containing Dam sites, DnaA-ATP-to-DnaA-ADP ratios might influence the importance of sequestration. Consequently, systems based on the quality-quantity control of the initiator (e.g., the ratio of DnaA-ATP:DnaA-ADP) or of the targets of the initiator (e.g., of the methylation state of DnaA boxes) might co-occur in vivo to different degrees.  
However, an important question remains as to how sequestration ends. One simple explanation is a spontaneous SeqA detachment on the basis of the equilibrium constant: a SeqA-Dam M+/ - complex may be dissociated due to the accumulation of free Dam M+/ - sites produced by the progression of the replication fork. Another possibility is the disappearance of the hyperstructure involving SeqA and other hemimethylated DNA-binding proteins. [PERSON]'s group, for example, reported that in _B. subtilis_, which lacks Dam methylation, a 270 kD complex exists that is associated with the membrane and that involves the YabA protein, which negatively regulates DnaA [84].