SeqA-MEDIATED HEMIMETHYLATION AND SEQUESTRATION
In some types of bacteria, including E. coli, there is yet another means of delaying the initiation of new rounds of chromosome replication. As with the mechanisms described above, replication itself plays a role in this regulatory pathway where methylation of the DNA helps delay initiation. In E. coli and other enteric bacteria, the A's in the symmetric sequence GATC/CTAG are methylated at the N6 position. These methyl groups are added to the bases by the enzyme deoxyadenosine methylase (Dam or Dam methylase), but this occurs only after the nucleotides have been incorporated into the DNA. Since DNA replicates by a semiconservative mechanism, the A in the GATC/CTAG sequence in the newly synthesized strand remains temporarily unmethylated after replication of a region containing this sequence (Figure 1.24). The DNA at this site is said to be hemimethylated if the bases on only one strand are methylated.
The hemimethylated state is important in the context of regulation of initiation because a trans-acting protein called SeqA only binds with high affinity to hemimethylated GATC/CTAG sequences. SeqA is an essential facilitator of hemimethylation control of replication initiation and is found only in bacteria that have Dam. The sequence GATC/CTAG is found 11 times within oriC, much more often than would be expected by chance alone (Figure 1.15). GATC/CTAG sequences are also associated with the low-affinity DnaA-binding sites across the origin. Furthermore, the promoter region of the dnaA gene, the region in which mRNA synthesis initiates for the DnaA protein, also has GATC/CTAG sequences. SeqA is able to bind all of these strategically located GATC/CTAG sites after they are replicated (and therefore rendered hemimethylated), which has the effect of delaying the conversion to full methylation at these sites for about one-third of a cell cycle. SeqA also blocks binding of DnaA to the low-affinity sites and inhibits expression of the dnaA gene. SeqA bound to hemimethylated DNA may associate with the cell membrane to sequester the oriC region after initiation of DNA replication (Figure 1.25). Sequestration of the hemimethylated oriC region is predicted to render it temporarily nonfunctional for the initiation of a new round of replication and delays its methylation by the Dam methylase (see Slater et al., Suggested Reading).
Figure 1.24 Replication creates hemimethylated DNA. (A) The A in the sequence GATC is methylated on both strands (Am and mA). (B) After replication, the A in GATC in the new strand is not immediately methylated by the Dam methylase. (C) Eventually, GATC sites in the new strand are methylated, converting the DNA back to the fully methylated state.
Figure 1.25 Model showing the possible functional consequences of SeqA binding with regularly and closely spaced GATC sites in the oriC region and broadly and irregularly spaced GATC sites outside the oriC region in the E. coli chromosome. Methylated GATC sites in a strand are represented by orange circles. Unmethylated GATC sites in a strand are represented by blue circles. (A) Before initiation, the oriC region is methylated in both strands. (B) After initiation, only one of the two strands is methylated; hence, the region is hemimethylated. The newly synthesized strand is shown in a lighter shade of blue. (C) SeqA binds the regularly spaced hemimethylated GATC sites in oriC and is able to interact with the membrane, thereby sequestering oriC and preventing further initiation and drastically slowing methylation. (D) Outside of the oriC region, where GATC sites are not regularly spaced and are situated farther apart, SeqA may be a bridge between the two hemimethylated strands to help coordinate processing events important for facilitating repair and recombination.
SeqA activities outside of oriC
In addition to the GATC/CTAG sequences associated with oriC and the dnaA promoter, SeqA also interacts with the GATC/CTAG sequences as the replication forks progress around the chromosome, effectively marking the location of the replisome (Figure 1.25). SeqA bound to transiently hemimethylated GATC/CTAG sites immediately behind the DNA replication forks may be capable of bringing together the nascent sister chromosomes, and it also appears to negatively regulate the decatenation activity of topoisomerase (see Joshi et al., Suggested Reading). Both of these processes may mediate a form of sister chromosome cohesion that in turn may help to regulate processing of the new DNAs by positioning them for DNA repair and recombination. Interestingly, SeqA is essential in mutants that lack the major pathways of DNA recombination involving RecA, supporting an additional role in protecting genome integrity. Additionally, chromosomes appear to be vulnerable to many types of mobile DNA elements during DNA replication, especially when replication forks stall (see Fricker and Peters, Suggested Reading). SeqA-facilitated processes may help to protect DNA replication forks from mobile elements as one of multiple mechanisms of protecting genome integrity.
The Bacterial Nucleoid
The nucleoid was described with respect to chromosome segregation above. Indeed, experiments in many of the model systems indicate that bacteria carefully coordinate the position of the chromosome in the cell. Through techniques in which individual positions in the chromosome can be localized in whole cells, it has been shown that genes are located in the chromosome in roughly the same order as one would presume by looking at the DNA sequence. A variety of techniques are providing insight into how the structure of the chromosome is maintained in the cell. The molecular mechanisms that maintain the chromosome structure remain a mystery, but specific systems are likely to exist to ensure that the chromosome is available for transcription, recombination, and other functions.
Supercoiling in the Nucleoid
Supercoiling is one of the mechanisms that help compact and organize the chromosome. Supercoiling also affects the expression level of many genes. However, supercoiling will be lost if one of the strands of the DNA is cut, thereby allowing the strands to rotate around each other. The phosphodiester bond connecting the two deoxyribose sugars on the other strand serves as a swivel and rotates, resulting in relaxed (i.e., not supercoiled) DNA. A DNA with a phosphodiester bond broken in one of the two strands is said to be nicked. A variety of experiments suggest that the nucleoid is packaged in subregions that constrain supercoiling. Topological barriers would prevent the entire chromosome from losing supercoiling when there is a nick in the genome. Figure 1.26 illustrates supercoiling.