Prokaryotic DNA Replication

Prokaryotic DNA Replication

• Replication of DNA is the process by which an organism creates an exact copy of its DNA so that it can be passed onto offspring.
• Before a cell splits, its DNA must be replicated to create an exact copy of the parent’s DNA in the daughter cell.
• When DNA replicates, one of its strands becomes the new daughter strand, and the other remains the parent strand. This is a semi-conservative process.
• DNA replication in bacteria is a well-studied phenomenon thanks to the prevalence of E. coli as a model organism, but the process is very similar across all prokaryotes.
• There is just one place from which replication can begin, and it happens in both directions (OriC).
• occurs in the cytoplasm of a cell.
• Only in the direction from 5′ to 3′ is synthesis possible.
• Both a leading and a trailing strand of DNA are created throughout the manufacturing process.
• Okazaki fragments, which are little pieces of DNA, are made and used to create lagging strands.

Mechanism of prokaryotic dna replication

The synthesis of a DNA molecule can be divided into three stages: 

1. Initiation
2. Elongation
3. Termination

distinguished both by the reactions taking place and by the enzymes required. As you will find here and in the next two chapters, synthesis of the major information containing biological polymers-DNAs, RNAs, and proteins-can be understood in terms of these same three stages, with the stages of each pathway having unique characteristics. The events described below reflect information derived primarily from in vitro experiments using purified E col’i proteins, although the principles are highly conserved in all replication systems.

Enzymes involved in prokaryotic dna replication

• Helicases: To begin replication, helicases unwind the DNA helix.
• SSB proteins: Proteins belonging to the SSB family bind to unwound DNA strands, stopping the DNA helix from reforming.
• Primase: To begin making DNA, primase first creates an RNA primer.
• DNA Polymerase III (DNAP III): Extends DNA strand by joining the 3′ ends of two complementary strands of deoxyribonucleotides. DNAP III restricts synthesis to the 5′-to-3′-direction.
• DNA Polymerase I (DNAP I): RNA primer is swapped out for the correct deoxynucleotides by DNA Polymerase I (DNAP I).
• DNA topoisomerase I: It creates a nick in one strand of DNA, which relaxes the DNA helix during replication.
• DNA topoisomerase II: forms supercoils in the helix by making nicks in both strands of DNA, which reduces stress on the DNA helix during replication.
• DNA ligase: DNA ligase is an enzyme that joins together two pieces of DNA by forming a 3′-5′ phosphodiester link.

Initiation of Prokaryotic DNA Replication

Protein required for initiation of Replication in Prokaryotes

Step 1: Oric

• E. coli’s replication origin, called oriC, is 245 base pairs long and shares DNA sequence components with other bacteria’s replication origins.
• Figure depicts the overall structure of the conserved sequences.
• Two types of sequences are of particular interest: five repeats of a 9-bp sequence (R sites) that serve as binding sites for the key initiator protein DnaA, and a region rich in A:T base pairs called the DNA unwinding element (DUE).
• There are other binding sites for the proteins IHF (integration host factor) and FIS in addition to the three DnaA-binding sites (I sites) (factor for inversion stimulation).
• Another DNA-binding protein that is involved but does not have a specific binding site is HU (a histonelike bacterial protein originally named factor U).

Step 2: Oric Recognize by DnaA

• During the beginning phase of replication, at least 10 different enzymes or proteins are involved. They open the DNA helix at the beginning and set up a complex that will be used in the next steps.
• The DnaA protein, which is part of the AAA+ ATPase protein family, is the most important part of the initiation process (ATPases associated with diverse cellular activities). DnaA is a AAA+ ATPase that forms oligomers and breaks down ATP at a slow rate.
• This ATP hydrolysis acts as a switch that lets the protein change between two different states.
• The ATP-bound form of DnaA is active, while the ADP-bound form is not.
• Eight ATP-bound DnaA protein molecules come together to form a helix that wraps around the R and I sites in ori,C.
• DnaA is more likely to bind to R sites than I sites, and it can bind to R sites just as well when it is bound to ATP or ADP.
• The I sites, which only bind to DnaA that is bound to ATP, make it possible to tell the difference between active and inactive DnaA.
• The tight wrapping of the DNA around this complex in a right-handed way makes a positive supercoil that works well. The strain in the nearby DNA makes the A=T-rich DUE region become less stable.
• The complex that forms at the replication origin also has Hu, IHR, and FIS, which are DNA-binding proteins that help DNA bend.

Step 3: DnaC Protein

• The DnaC protein, which is also a AAA+ ATPase, then binds the DnaB protein to the two separate strands of DNA in the denatured area.
• The ring-shaped DnaB helicase forms a tight complex with six DnaC subunits, each of which is bound to ATP.
• This interaction between DnaC and DnaB opens the DnaB ring. Another interaction between DnaB and DnaA helps this process along.
• One of the ring-shaped DnaB hexamers is put on each strand of DNA in the DUE.
• The ATP that is bound to DnaC is broken down by water, which frees DnaC but leaves DnaB bound to the DNA.

Step 4: DnaB Protein, SSB, DNA gyrase

• Loading the DnaB helicase is the most important step in the beginning of replication.
• As a replicative helicase, DnaB moves along single-stranded DNA in the 5′-to-3′ direction, unwinding the DNA as it goes.
• So, the DnaB helicases that are attached to the two DNA strands move in opposite directions. This makes it possible for two replication forks to form.
• All of the other proteins at the replication fork are connected to DnaB in some way.
• The r subunits link the DNA polymerase III holoenzyme together. Below, we’ll talk about other DnaB interactions.
• As replication begins and the DNA strands separate at the fork, many molecules of single-stranded DNA-binding protein (SSB) bind to and stabilise the separated strands, and DNA gyrase (DNA topoisomerase II) relieves the topological stress caused by the unwinding reaction before the fork.
• The only part of DNA replication that is known to be controlled is the beginning, or initiation. It is controlled so that replication only happens once in each cell cycle.
• We don’t fully understand how regulation works yet, but genetic and biochemical studies have given us clues about several different ways regulation works.

Step 5: Protein Hda

• Once DNA polymerase III and the B subunits have been loaded onto the DNA, which marks the end of the initiation phase, the protein Hda binds to the B subunits and interacts with DnaA to speed up the hydrolysis of the ATP that is bound to DnaA.
• Hda is also a AAA+ ATPase that is very similar to DnaA. (its name is derived from homologous to DnaA).
• This breakdown of ATP causes the DnaA complex at the beginning of the cell to fall apart.
• The protein changes between its inactive (with bound ADP) and active (with bound ATP) forms every 20 to 40 minutes. This happens because DnaA slowly lets go of ADP and rebinds ATP.

Step 6: DNA adenine methylation

• Methylation of DNA and interactions with the bacterial plasma membrane can change when replication starts.
• The Dam methylase methylates the N6 spot of adenine in the palindromic sequence (5′)GATC, which is part of the ori,C DNA. (DNA adenine methylation is what “Dam” means in biochemistry.)
• The E. coli ori,C region has a lot of GATC sequences. It has 11 of them in 245 bp, while the average number of GATC sequences in the whole E. coli chromosome is 1 in 256 bp.
• The DNA is hemimethylated right after replication. The parent strands have methylated ori,C sequences, but the new strands don’t. The protein SeqA now binds to the hemimethylated oriC sequences and keeps them from moving. This is done by the interaction with the plasma membrane (how this happens is not known).
• After some time, ori,C is released from the plasma membrane, SeqA breaks apart, and the DNA must be fully methylated by Dam methylase before it can bind to DnaA again and start a new round of replication.

Elongation of Prokaryotic DNA Replication

• During the elongation phase of replication, Ieading strand synthesis and Lagging strand synthesis take place. These are two separate but related processes.
• At the replication fork, there are several enzymes that are important for making both strands. First, DNA helicases unwind the parent DNA. Then, topoisomerases relieve the topological stress that is caused.
• Then, SSB is used to keep each strand in place. Synthesis of the leading and lagging strands is very different from this point on.

Leading strand synthesis

• The simpler of the two processes, leading strand synthesis starts with primase (DnaG protein) making a short (10–60 nucleotide) RNA primer at the replication origin.
• DnaG and DnaB helicase work together to make this reaction happen. The primer is made in the opposite direction that the DnaB helicase is moving.
• In reality, the DnaB helicase moves along the strand that becomes the lagging strand during DNA synthesis. However, the first primer that is laid down during the first DnaGDnaB interaction starts DNA synthesis on the leading strand in the opposite direction.
• A DNA polymerase III complex linked to the DnaB helicase on the other DNA strand adds deoxyribonucleotides to this primer.
• Then, the process of making the leading strand keeps going at the same rate as the unwinding of DNA at the replication fork.

Lagging strand synthesis

• We’ve already said that lagging strand sythesis is done in short Okazaki fragments.
• First, primase makes an RNA primer. Then, just like in leading strand synthesis, DNA polymerase III binds to the RNA primer and adds deoxyribonucleotides.
• On this level, putting together each Okazaki fragment seems easy, but it’s actually quite hard. The coordination of leading and lagging strand synthesis is what makes it hard.
• A single asymmetric DNA polymerase III dimer makes both strands. This is done by looping the DNA of the Lagging strand, which brings the two points of polymerization together.

Read Also: what is a major difference between eukaryotic dna replication and prokaryotic dna replication?

Synthesis of Okazaki fragment

• Elegant enzymatic choreography is required for the formation of Okazaki fragments on the lagging strand.
• Primosomes are functional units inside the replication complex that consist of DnaB helicase and DnaG primase.
• Core polymerase subunits of DNA polymerase III are always at work synthesising the leading strand, while core polymerase subunits of DNA polymerase III cycle from Okazaki fragment to fragment on the looped lagging strand.
• As DNA polymerase III moves along the lagging strand template in the 5′-3′ direction, the DnaB helicase tethered in front of it unwinds the DNA at the replication fork.
• There are cases where DnaG primase joins forces with DnaB helicase to produce a short RNA primer.
• After that, the DNA polrmerase III clamp Loading complex moves a fresh β sliding clamp to the primer.
• At the end of Okazaki fragment synthesis, DNA polymerase III core subunits detach from the β sliding clamp (and the finished Okazaki fragment) and re-associate with the new clamp, signalling the end of replication.
• A new Okazaki fragment will be created as a result of this. A replication fork contains a complex of proteins called the replisome, which is important for coordinating DNA synthesis at the fork.

The clamp Loading complex of DNA polymerase III

• The clamp Loading complex of DNA polymerase III, consisting of parts of the two τ subunits along with the γ, δ, and δ’ subunits, is also an AAA+ ATPase.
• Together, ATP and the novel B sliding clamp form a stable combination. The dimeric clamp undergoes strain as a result of the binding, and the ring opens up at one of the subunit interfaces.
• Through this rip, the newly primed lagging strand is introduced to the ring.
• Hydrolysis of ATP by the clamp loader then allows the B sliding clamp to close around the DNA.

DNA synthesis

• The replisome is responsible for the rapid synthesis of DNA, at a rate of -1,000 nucleotides per second per strand (leading and lagging).
• DNA ligase seals the residual nick in an Okazaki fragment after DNA polymerase I has removed the RNA primer.
• DNA ligase is an enzyme that catalyses the creation of a phosphodiester link between a 3′ hydroxyl and a 5′ phosphate on opposite ends of DNA strands.
• Adenylylation is required to activate the phosphate. The ATP is used by DNA ligases that have been purified from viruses and eukaryotes.
• Bacterial DNA ligases are distinct in that many of them derive the AMP activating group from the coenzyme NAD+, which typically catalyses hydride transfer events. DNA ligase is another DNA metabolic enzyme that has emerged as a crucial tool in recombinant DNA research.

Proteins required for replisome

Termination of Prokaryotic DNA Replication

• Ultimately, the circular DNA replication split in two. The two halves of an E. coli chromosome join at a terminus region called Ter, which contains multiple copies of a 20-base-pair sequence.
• The Ter sequences on the chromosome are organised in a way that traps a replication fork inside.
• Protein Tus binds to specific sequences in the Ter RNA (terminus utilisation substance).
• Only one way of a replication fork can be stopped by the Tus-Ter complex. Each replication cycle only allows one Ttrs-Ter complex to work, and that is the complex that is encountered first by one of the two replication forks. Ter sequences may prevent over replication by one fork in the case that the other is delayed or halted by encountering DNA damage or another impediment, even though opposing replication forks typically halt when they intersect.
• So, replication stops when one of the forks runs into a working Tus-Ter complex, and the other fork stops when it runs into the first (arrested) fork.
• Following replication of the last few hundred base pairs of DNA (by a mechanism we don’t fully understand), two topologically connected (catenated) circular chromosomes are formed.
• Catenanes are the name given to interconnected DNA cyclins.
• The enzyme topoisomerase IV is necessary for the separation of the catenated rings in E. coLi (a type II topoisomerase).
• Once the chromosomes have been untangled, they are distributed evenly throughout the two daughter cells.
• Many DNA viruses that infect eukaryotic cells undergo a similar process near the end of their replication cycle, which is also characteristic of other circular chromosomes.

Role of topoisomerases in replication termination

• The finished chromosomes are united as catenanes, or topologically interlinked circles, as a result of replication of the DNA separating opposing replication forks.
• The circles are not chemically bonded to one another, but they are impossible to disentangle without the help of enzymes called topoisomerases.
• DNA topoisomerase lV, a type ll topoisomerase in E. coli, is responsible for transiently breaking both DNA strands of one chromosome and allowing the other chromosome to pass through the breach, therefore separating catenated chromosomes.

Other Prokaryotic replication models

The duplication of theta type has already been mentioned. Rolling-circle replication and D-loop replication are two further forms of bacterial reproduction.

Rolling Circle Replication

• The same circular template DNA spins during bacterial conjugation, and it is around this DNA that a new strand of DNA forms.
• The relaxase enzyme forms a nick in one strand of the conjugative plasmid at the oriT when signalling initiates conjugation.
• Relaxase can function either independently or as part of a larger complex of over a dozen proteins called a relaxosome.
• Relaxase enzyme TraI, along with its cofactors TraY and TraM, and the integrated host factor IHF, make up the relaxosome of the F-plasmid system.
• Following its nicking, the T-strand is unravelled from the intact strand and transmitted to the recipient cell in a 5′-terminus-to-3′-terminus manner.
• In either case, the residual strand undergoes replication, with or without the help of conjugation (conjugative replication similar to the rolling circle replication of lambda phage).
• It’s possible that a second nick is necessary for effective transfer during conjugative replication. The second nicking event is supposedly blocked by compounds that imitate an intermediate step of the process, according to a recent paper.

D-loop replication

• Organellar DNA is the most common type of DNA to undergo D-loop replication, which results in the formation of a displacement loop, a triple stranded structure.

Describe The Three-step mechanism of DNA ligation

1. DNA ligase I is a cellular enzyme that catalyses the covalent ligation of adenosine monophosphate (AMP) to DNA via hydrolysis of adenosine triphosphate (ATP).
2. Following a nick in duplex DNA, the ligase polypeptide transfers an AMP group to the nick’s 5′ phosphate termini.
3. In a process involving nucleophilic assault by the 3’HO group and release of AMP, the non-adenylated ligase catalyzes the creation of the phosphodiester link.

Significance

• Replication of DNA is a crucial genetic mechanism required for cell proliferation and expansion.
• The process of DNA replication results in the production of a new molecule of the essential nucleic acid DNA.
• DNA replication plays a crucial role in controlling how cells divide and expand.
• The entire genome is preserved for future generations.

FAQ

References

• Howes, Timothy & Tomkinson, Alan. (2012). DNA ligase I, the replicative DNA ligase. Sub-cellular biochemistry. 62. 327-41. 10.1007/978-94-007-4572-8_17. 
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