E. coli and S. enterica must duplicate their circular genomes, which each contains around 4.7 megabase pairs of double-stranded DNA, before splitting into two daughter cells. The nutritional state of the cell and the mechanism that initiates the commencement of new DNA polymerization must be perfectly coordinated for this important step of cellular reproduction to be completed successfully. It has recently been discovered that complex regulatory circuits direct the assembly of a multicomponent molecular machine (the orisome) that separates DNA strands and loads replicative helicase at oriC, the unique chromosomal origin of replication, to control the initiation step of DNA replication.
The orisome must be dismantled and its components inactivated after the replisome has been constructed and fresh replication forks have been passed to ensure that only one new round of DNA synthesis is triggered from each replication origin throughout a cell division cycle.
Although many parts of the process remain unknown, we will review current efforts to better understand the controlled protein-DNA interactions that are important for properly timed chromosomal replication initiation in this chapter. The unique structural and biochemical properties of the bacterial initiator protein DnaA, as well as recently found nucleotide sequence features inside E. coli oriC, will be discussed. We’ll also talk about the coordinated mechanisms that keep DNA replication from happening at the wrong time.
History of Initiation of replication
When bacteria develop quickly, they have more DNA, and when they grow slowly, they have less, with the amount of DNA per cell fluctuating continually with the growth rate. This finding, made by Schaechter in 1958, highlighted doubts regarding how growth rate-regulated expansion and contraction of DNA content were accomplished. Although it might seem fair to assume that replication forks would simply move quicker and faster, this is not the case.
E. coli that is exponentially proliferating will always divide 60 minutes following the start of each round of chromosomal DNA synthesis. The time it takes to prepare for a cycle of chromosomal replication, on the other hand, is not constant and is entirely dependent on the rate of cellular growth.
Because replication forks on Escherichia coli and Salmonella chromosomes continue bidirectionally from fixed replication sources, newly separated daughter cells will inherit dichotomously branched chromosomes (dubbed theta structures after the Greek letter theta).
During the cell division cycle, however, all copies start replication at the same time.
The I+ C + D rule explains why quickly growing bacteria have more DNA and focuses emphasis on the mechanism that initiates DNA synthesis, a critical regulatory phase in the bacterial cell cycle.
Key Points
- Initiator proteins recognise and bind to the replicator, allowing DNA replication to begin. Initiator proteins perform a variety of functions, including recognition of the ori and recruitment of replication factors, as well as melting of double-stranded DNA and replicative DNA-helicase activity.
- Initiator proteins have a surprising property: they bind DNA with low sequence specificity, even though identifying the initiation site is thought to require great specificity. The lack of specificity in viral initiator proteins is due to the presence of a nonspecific DNA-binding activity that is essential for the melting and unwinding stages. Nonspecific binding activity is inhibited, resulting in highly specific binding for ori identification.
- DNA-binding domains, which direct initiator proteins’ site-specific DNA binding, are structurally related in initiators from diverse virus groups and show that these domains have a common ancestor with proteins that bind RNA.
- Viral initiator proteins assemble in an orderly form into various complexes that provide sequence-specific recognition, DNA-melting activity, and DNA-helicase activity in succession, utilising multiple protein–DNA and protein-protein interactions. This ‘hardwiring’ of the subsequent initiator activities enables a highly efficient and reliable initiation procedure.
- A replication origin is unwound and two molecules of replicative DNA helicase are loaded onto accessible single-strands before bidirectional replication forks are assembled. Three proteins, DnaA, DnaB, and DnaC, are adequate in Escherichia coli and Salmonella to carry out these functions. DnaA aids DnaC, a specialised helicase loading protein, in putting DnaB, the replicative DNA helicase, onto accessible single-stranded DNA by unwinding oriC at specified sites.
Initiation Gene Expression
Gene expression begins with transcription. A gene’s DNA sequence gets transcribed into RNA during this procedure.
The double helix of DNA must unravel near the gene being transcribed before transcription can take place. A transcription bubble is a section of DNA that has opened up.
The template strand is one of the two exposed DNA strands that is used as a template during transcription. The RNA product complements the template strand and is nearly identical to the nontemplate (or coding) strand of DNA. There is one significant difference, however: all T nucleotides are substituted with U nucleotides in newly synthesised RNA.
The +1 site, also known as the initiation site, is the DNA sequence from which the first RNA nucleotide is transcribed. Negative values are assigned to nucleotides that come before the initiation site and are referred to as upstream nucleotides. Positive values indicate downstream nucleotides, which are those that come after the start site.
If the transcribed gene codes for a protein (which many do), the RNA molecule will be read and translated into a protein.
Conclusion
The main characteristics of DNA replication start are remarkably similar across eukaryotic species, including plants. These characteristics include chromatin structure as multiple replicons, pre-replication complexes (pre-RCs) assembly at replication origins, cell cycle kinase activation of pre-RCs, and replisome assembly.
The differences between distinct eukaryotic groupings are found in the higher-level regulatory mechanisms that are linked to the organisms’ lifestyles. Thus, there is convincing evidence that hormones involved in many aspects of growth and development operate on, among other things, the commencement of DNA replication in higher plants.