1. Introduction
When growing cells divide, they need to copy their genetic material and distribute it to ensure that each daughter cell receives one copy. This is a challenging task especially when the enormous length of the DNA compared to the cell size is considered. During DNA replication, organization of the chromosomes is even more demanding, since replication forks continuously produce new DNA. This DNA contains all the information required to build the cells and tissues of a prokaryotic or an eukaryotic organism. The exact replication of this information in any species assures its genetic continuity from generation to generation and is critical to the normal development of an individual. The information stored in DNA is arranged in hereditary units known as genes that control the identifiable traits of an organism. Discovery of the structure of DNA and subsequent elucidation of how DNA directs synthesis of RNA, which then directs assembly of proteins -the so-called central dogma - were monumental achievements that marked the early days of molecular biology. However, the simplified representation of the central dogma as DNA → RNA → protein does not reflect the role of proteins in the synthesis of nucleic acids. Moreover, proteins are largely responsible for regulating DNA replication and gene expression, the entire process whereby the information encoded in DNA is decoded into the proteins that characterize various cell types. Two of these proteins are the DNA adenine methyltransferase (Dam) and the DNA-Binding Protein (SeqA).
1.1. The Dam methyltransferase
Methylation of DNA by the Dam methyltransferase provides an epigenetic signal that influences and regulates numerous physiological processes in the bacterial cell, including chromosome replication, mismatch repair, transposition, and transcription. A growing number of reports ascribed a role to DNA adenine methylation in regulating the mechanisms of DNA replication in diverse pathogens like in
The natural substrate for the enzyme is hemimethylated DNA, where one strand is methylated and the other is not. This is the configuration of DNA immediately behind the replication fork. Double stranded DNA is a better methyl receiver than denatured DNA, and there is little difference in the rate of methylation between unmethylated and hemimethylated DNA [28]. The Dam enzyme appears to have two SAM binding sites; one is the catalytic site and the other increases specific binding to DNA, probably through an allosteric transformation [13]. Dam is thought to bind the template and to slide processing along the DNA, methylating about 55 GATC sites per binding event [60]. There are about 130 molecules of Dam per
Dam competes with two other proteins, MutH and SeqA, for hemimethylated GATC substrate sites. These two proteins act before Dam to participate in the removal of replication errors (MutH) and to form the compact and properly super coiled chromosome structure for the nucleoid (SeqA). Increasing the cellular level of Dam causes a decrease in the amount of hemimethylated DNA, and prevents these two proteins from carrying out their functions, leading to an increased mutation rate and a change in the super coiling of the chromosome, respectively [27; 42; 40]. Although Dam methylase is a highly processing enzyme, it may become less processing at GATC sites, flanked by specific DNA sequences [50]. Reduced rate of processing may allow for a competition between Dam and specific DNA- binding proteins, thus permitting the formation of non-methylated GATCs which depended on the growth phase and the growth rate, suggesting that the proteins that bind to them could be involved in gene expression or in the maintenance of chromosome structure. The unmethylated dam sites appear to be mostly [53] or completely [49] modified in strains overproducing Dam, suggesting that the enzyme competes with other DNA - binding proteins at these specific sites. In addition to the unmethylated GATC sites discussed above, persistent hemimethylated sequences have been detected in the chromosome [48; 18]. These are distinct from the transiently hemimethylated GATC sites that occur immediately behind the replication fork due to the time lag between DNA replication and Dam methylation.
1.2. The SeqA protein
SeqA protein was discovered in some prokaryotes as a protein involved in the methylation / hemimethylation cycle of DNA replication [41]. This protein regulates the activation of the chromosome replication origin [41]. Experiments have shown that SeqA has a high affinity to hemimethylated as compared to fully methylated DNA. It binds specifically to GATC sequences which are methylated on the Adenine of the old strand but not on the new strand. Such hemimethylated DNA is produced by progression of the replication forks and lasts until Dam methyltransferase methylates the new strand (Figure 2). It is therefore believed that a region of hemimethylated DNA covered by SeqA follows the replication fork.
Proper chromosome segregation also requires SeqA [5]. Furthermore, SeqA trails the DNA replication fork and may contribute to nucleic organization in newly replicated DNA [17; 37; 40; 67]. Aside from its roles in chromosome replication and nucleic segregation, SeqA is known to regulate the transcription of certain genes. In bacteriophage lambda, SeqA activates the
1.3. The competition of Dam methylase and SeqA for GATC sites
Newly synthesized hemimethylated DNA is also a target for Dam methylase. In fact, Dam and SeqA have been suggested to be in competition for hemimethylated DNA [34]. Experiments with unsynchronized cells indicate that the sequestration period becomes shorter upon Dam over-expression [15; 57]. SeqA binding is largely limited to hemimethylated DNA, and the action of Dam will, therefore, transform DNA into a non-target for SeqA. This protein was able to bind DNA despite Dam overproduction. Recent findings showed that SeqA bound to DNA was not actively dissociated by Dam methylase [34]. The same study showed that the SeqA protein spontaneously dissociated from bound DNA after some minutes
2. DNA replication
The initiation of chromosomal replication occurs only once during the cell cycle in both prokaryotes and eukaryotes. This initiation is the first and tightly controlled step of a DNA synthesis. Because much of what is known about the regulation of the initiation of bacterial chromosomal replication comes from studies of
In prokaryotic cells, DNA replication and segregation are not temporally separated processes. Some evidence suggests that newly synthesized DNA is continuously segregated to opposite cellular positions [45; 52]. Other work indicates that some parts of segregation may be more abrupt and domain specific [7; 21]. Coordination of DNA replication and chromosome segregation is complicated by the ability of growing with overlapping replication cycles [19; 55]. Whereas during slow growth, chromosomes are replicated in a simple pattern with only one pair of forks; replication during fast growth occurs with one pair of old and two pairs of new forks on one chromosome. (Forks are considered to be ‘old forks’ as soon as new forks appear at initiation.) Depending on the exact conditions, a cell can have four copies of the multi-fork chromosome and a total of 24 replication forks per cell [22; 44]. How the cell meets the obvious need for efficient organization during such extensive replication is largely unknown. However, the SeqA protein is one of the strongest candidates to contribute [41; 61]. Loss of SeqA leads to severe growth impairment during the rapid but not slow growth [16]. Biochemical studies established that SeqA binding is specific to the sequence GATC with high preference for hemimethylated over fully methylated DNA [10; 58; 11; 34]. Hemimethylation occurs at newly replicated GATC sites which have not yet been re-methylated by the Dam methylase. A transient hemimethylation after the passage of the replication fork was found in an analysis of 10 individual GATC sites [18]. Similarly, transient binding of SeqA was detected at seven genomic sites with multiple GATC sequences [67]. Multiple DNA- bound SeqA dimers can oligomerize to form a higher order structure [25; 47]. The above findings suggested that a SeqA complex follows the replication forks, potentially in a tread milling fashion, growing at the leading end and diminishing at the tailing end. The reduction of the SeqA bound region at the most replisome-distant GATCs would come about through the activity of Dam which turns these sites into non-targets for SeqA by its methylation activity. The process described above is called DNA sequestration.
3. DNA methylation by Dam protein
As mentioned above, the Dam methyltransferase of
4. DNA sequestration by SeqA protein
Replication of the bacterial chromosomal DNA initiates only once, at a specific region known as the origin of chromosomal replication (
The chromosomal DNA is methylated at adenine residues in GATC sequences by Dam methylase. Following passage of the DNA replication fork, GATC sites methylated on the top and bottom strands in a mother cell (denoted as fully methylated) are converted into two hemimethylated DNA duplexes: one methylated on the top strand and non-methylated on the bottom strand and one methylated on the bottom strand and non-methylated on the top strand due to semi-conservative replication. Most GATC sites are rapidly re-methylated by the enzyme Dam methylase and exist in the hemimethylated state for only a fraction of the cell cycle (Figure 3).
Exceptions to this are the DNA replication origin of
In the case of
4.1. Binding of SeqA to Hemimethylated GATC Sequences
As mentioned above, the adenine residues of GATC sequences are methylated on their 6 amino group by Dam methyltransferase [23; 18]. Upon replication, the GATC sequences on the newly replicated strand remain transiently unmethylated, leading to a hemimethylated state of the DNA duplex. The new strand is subsequently methylated by Dam, and the duplex becomes methylated on both strands. The initiation of chromosome replication at the origin of chromosomal replication
Topoisomerase IV is essential for the de-catenation and segregation of replicated chromosomes at cell division [35; 36; 1; 68; 29]. Together with DNA gyrase, it also removes the positive super coils that accumulate in front of replication forks and growing mRNA transcripts. SeqA has been shown to promote the relaxation and the de-catenation activity of topoisomerase IV [33]. This appears to result from a specific interaction between topoisomerase IV and SeqA. Besides the asynchrony and overinitiation of chromosomal replication,
The C-terminal region of SeqA interacts via hydrogen bonds and van der Waals contacts with the major groove of DNA, with the hemimethylated A-T base pair and also with the surrounding bases and DNA backbone. The Nuclear Magnetic Resonance (NMR) structure of hemimethylated GATC revealed that it has an unusual backbone structure and a remarkably narrow major groove and suggested that this peculiar structural feature might contribute to recognition of hemimethylated GATC sites by SeqA protein [6]. To form a stable SeqA-DNA complex in the presence of competitor DNA, one SeqA tetramer binds to each of two hemimethylated GATC sequences [26] that are up to 31 bases apart on the DNA [17]. The sequential binding of SeqA tetramers to hemimethylated sites leads to the formation of higher order complexes [26]. Further, the binding of SeqA proteins to at least six adjacent hemimethylated sites induces the aggregation of free proteins onto the bound proteins, thus implying cooperative interaction between the SeqA proteins.
4.2. Effects of seqA disruption on DNA replication
As we said before, following the replication fork progression and the nascent strand synthesis, the daughter DNA becomes hemimethylated. SeqA protein binds to the hemimethylated GATC sequences (hemi-sites) and performs various roles to control the cell cycle progression. Immediately after the initiation of replication SeqA binds to the replicated
When rounds of replication are allowed to run to completion, the number of chromosomes per cell is 2n (n = 0, 1, 2, 3, etc). When initiations are asynchronous, as in
So, the flow cytometer analysis of a
This loss of synchrony affected culture growth rates and cell size distributions only slightly and suggest that
The OFF to ON phase rate was reduced in a
5. Membrane sequestration hemimethylated of oriC
The coordination of the synchronization of the replication initiation, the activation of the DnaA protein at
Prior to initiation of DNA replication, Dam methylase sites are fully methylated. Immediately following replication, the newly synthesized strand is unmethylated, and the resulting hemimethylated origin is sequestered at the lipid bilayer of membrane by SeqA. This is not accessible to replicatively active ATP–DnaA. After approximately one-third of the cell cycle, the sequestered origin is released and methylated by Dam methylase. At this point in the cell cycle, the levels of ATP–DnaA are not sufficient to catalyze a new round of replication. As such, sequestration serves as a mechanism to prevent secondary initiations. Subsequent work identified SeqA protein to be an essential factor in the
6. Cooperation between “Dam” and “SeqA” in DNA Replication
In
In fast-growing
In
7. Conclusion
In the
8. Future research
Our knowledge on the effects of Dam and SeqA proteins in
Finally, the way in which Dam and SeqA participate clearly in the DNA replication is a critical question that deserves further investigation in the near future, and may be research studies will have to identify explanations.
Acknowledgments
The work presented in this chapter was supported by the Tunisian Ministry of Higher Education, Scientific Research, and Technology. It has been performed at the biochemistry and molecular biology laboratory which belongs to the Faculty of Sciences of Bizerta. Many people have in different ways supported me in my work and contributed to the completion of this study. I would especially like to express my gratitude to:
Dr. Francisco Ramos-Morales (Departamento de Genética, Facultad de Biologia, Universidad de Sevilla, Spain).
Miss BCHINI Khouloud for proofreading and correcting this manuscript.
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