Leading strand and lagging strand synthesis in E.coli (Elongation stage of DNA replication)
- After DNA is unwound and a primer has been added, DNA polymerase elongate the polynucleotide strand by catalyzing DNA polymerization.
- The elongation phase of replication includes two distinct but related operations: leading strand synthesis and lagging strand synthesis. Several enzymes at the replication fork play important role in the synthesis of both strands.
- The replisome promotes rapid DNA synthesis by adding ~1000 nucleotides/s to each strand (leading strand and Lagging strand).
The 2 strands of DNA are antiparallel in nature. So replication has to happen in different directions on the 2 strands. But the DNA-dependent DNA polymerases catalyze polymerization only in one direction (5’ to 3’ direction). The two nascent DNA strands being synthesized at each replicating fork are being extended in the same direction. This creates some additional complications at the replicating fork. As result of this DNA synthesis occurs differently in two strands.
Template strand with 3’ to 5’ polarity: DNA synthesis continuous (daughter strand name: Leading strand)
Template strand with 5’ to 3’ polarity: DNA synthesis is discontinuous (daughter strand name: Lagging strand).
The DNA polymerase III holoenzyme synthesizes the leading and lagging strands simultaneously at the replication fork. So two unit of DNA polymerase III must be present at the replication fork, one for each strand.
Before entering to the topic you must remember this basic information on DNA polymerase holoenzyme
- We already discussed the structure of DNA polymerase III holoenzyme. Each holoenzyme consists of two copies of DNA Pol III core enzyme, two copies of beta sliding clamp and a copy of clamp loader (γ-complex).
- Each DNA Pol III core enzyme interacts with the γ-complex through τ protein (dimerizes the core enzyme). The τ protein consists of a flexible linker which allows DNA Pol III core enzymes to move independently. The δ subunit of the clamp loader interacts with the β sliding clamp.
- Coordination between lagging strand synthesis and leading strand synthesis is achieved by the dimerization of DNA polymerase enzymes at the replication fork.
- One of the catalytic cores of the DNA polymerase holoenzyme catalyses the synthesis of leading strand and the other catalytic core catalyses the synthesis of lagging strand.
Leading strand synthesis
- The leading strand is the strand of nascent DNA which is being synthesized in the same direction as the growing replication fork.
- The leading strand synthesizing DNA Pol III has an easy job because as the helicase separates the 2 strands, the leading strand constantly has a new template right in front of it.
- DNA polymerase III begins the synthesis of the leading strand by using the RNA primer (10 to 60 nucleotides) formed by primase (DnaG protein).
- Leading strand synthesis can proceed continuously from a single RNA primer in the 5’ to 3’ direction.
- As the replication fork moves, the DNA polymerase dimer moves with replication fork. Polymerase of the leading strand template remains attached to it and synthesizes the leading strand continuously. It does not release the template until replication has been completed.
Lagging strand synthesis
- The lagging strand is the strand of nascent DNA whose direction of synthesis is opposite to the direction of the growing replication fork. (So the lagging strand DNA Pol III is constantly finding newly separated template strand behind it, so it has to detach and find a starting place and work backwards compared to the leading strand).
- Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand. As a consequence, the DNA polymerase on this strand is seen to “lag behind” the other strand.
- During the elongation process, the primosome complex synthesis multiple RNA primers on the lagging strands at a distance of 1000 NTs
- DNA polymerase III core enzyme involved in lagging strand synthesis cycles from one Okazaki fragment to the next on the looped lagging strand during replication.
For the enzyme like DNA polymerase III it is highly unlikely to synthesize both leading and lagging strands moving in opposite direction. Two different models were proposed to explain the simultaneously synthesis of leading and lagging strands by a single DNA polymerase III complex.
1) Inverted loop model
- The DNA polymerase III forms an inverted loop in the lagging strand template by which the orientation of RNA primer will be reversed and its 3’-OH group will now face the replication fork.
- The DNA polymerase III moves in single direction, synthesizing the Okazaki fragments along with the leading strand.
- Once an Okazaki fragment is synthesized, the loop that is formed is released and the enzyme forms another inverted loop at the next RNA primer and this process continuous till the end of the DNA replication.
- (The lagging template strand is “looped” in order to invert the physical direction of synthesis, but not the biochemical direction)
2) Dragging model:
- The DNA polymerase III drags the lagging strand template towards inside through the active site and while dragging it synthesizes the Okazaki fragments and this process is repeated throughout the replication.
Coordination between the Leading and the Lagging Strands.
The looping of the template for the lagging strand enables a dimeric DNA polymerase III holoenzyme to synthesize both daughter strands simultaneously.
This is how actually replication fork of E.coli looks like. From the figure you can understand many things.
- The E.coli DNA replisome, which contains two DNA polymerase III holoenzyme complexes, synthesizes both the leading and the lagging strands. (The enzyme cores are shown in red color).
- As the helicase unwinds, the lagging strand template DNA is looped out and is bound by SSB proteins.
- RNA primase of the primosome complex synthesize new RNA primer at the fork junction to initiate new okazaki fragment synthesis.
- After initiation of an Okazaki fragment, the lagging strand core complex pulls the single stranded template through the β clamp while synthesizing the new strand.
Trombone model of Lagging strand DNA replication
- Changing the size of the loop formed by the lagging strand template during lagging strand synthesis. (Hence the name trombone model)
Figure 4: The trombone model for coordinating replication by two DNA polymerases at the E.coli replication fork
Note that in this model, leading strand synthesis is always ahead of lagging strand synthesis.
1) Initiation of an Okazaki fragment synthesis
- The DnaB helicase unwinds the DNA at the replication fork as it travels along the lagging strand template in the 5’ to 3’ direction.
(The DNA polymerase holoenzyme interacts with the DNA helicase through the τ subunits (which dimerizes the core DNA polymerases). This establishes a direct connection between the helicase-primase complex and the catalytic cores).
- As the helicase unwinds the DNA at the replication fork, the leading strand template is rapidly copied while the lagging strand template is looped out as single stranded DNA and are rapidly bound and stabilized by ssDNA binding proteins. This loop becomes larger as the unwinding point advances.
- DNA primase occasionally associates with DnaB helicase and synthesizes a short RNA primer for Okazaki fragment synthesis. Primer is extended by DNA polymerase III.
- Instead of moving independently down the lagging strand during synthesis, DNA polymerase III core has to pull back the lagging strand until it is done making DNA. So After initiation of an Okazaki fragment, the core polymerase III of holoenzyme involved in lagging strand synthesis pulls the single stranded template through the β clamp associated with it.
- The looping back of the lagging strand onto the replisome allows both leading- and lagging-strand DNA polymerases to synthesize in the same direction and facilitates recycling of the lagging-strand DNA polymerase by virtue of its proximity to the RNA primers newly synthesized at the fork.
2) Termination of Okazaki fragment synthesis
- The holoenzyme releases the lagging strand template when it encounters the previously synthesized Okazaki fragment. This possibly signals the primosome to initiate the synthesis of another lagging strand RNA primer.
3) Dissociation of core and beta clamp
- When the lagging strand DNA polymerase completes an Okazaki fragment, it is released from the sliding clamp and from the lagging strand template.
- The lagging strand polymerase remains at the fork through a direct linkage to the leading strand polymerase and helicase through τ (prevent falling off of the core polymerase)
4) Reassociation of beta clamp
- At intervals, primase synthesizes an RNA primer at an upstream position of the previously synthesized Okazaki fragment to prime new round of Okazaki fragment synthesis. After that they are dissociate from primosome complex.
- A new β sliding clamp is then positioned at the primer template junction by the clamp-loading complex of DNA polymerase III
- The primer – template junction with its associated sliding clamp again binds to the lagging strand DNA polymerase and it extends the RNA primer to form a new Okazaki fragment.
The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis.
- As each Okazaki fragment is synthesized, towards completion the loop gets smaller and smaller. The SSB-bound part of the loop would remain the same as SSB binds to the single strand to keep it untangled.
- Initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release.
Why the lagging strand template is looped out during DNA replication in E.coli?
During lagging strand synthesis, template DNA and newly synthesized lagging strand fragment loops out between the polymerase and the fork. This looping would allow the lagging-strand polymerase to move along with the rest of the replication fork instead of in the opposite direction. (Since both core polymerases are linked in the holoenzyme, they cannot move independently in opposite direction. If looping was not there one of the core polymerase has to wait until the other core completes DNA synthesis).
The DNA polymerase III forms an inverted loop in the lagging strand template by which the orientation of RNA primer will be reversed and its 3’-OH group will now face the replication fork. The looping and reverse processing has to occur over and over again during the synthesis of each Okazaki fragment.
Removal of RNA primers and formation of continous lagging strand
- Okazaki fragments are between 1000 and 2000 nucleotides long in prokaryotes. They are separated by ~120 nucleotide RNA primers and are unligated until RNA primers are removed.
- To form a continuous lagging strand of DNA, the RNA primers must eventually be removed from the Okazaki fragments and replaced with DNA.
The reaction proceeds in three steps: removal of the RNA primer, Filling the gap with deoxyribonucleotides and sealing of the adjacent DNA fragments
Step 1: In E. coli, RNA primers are removed by the combined action of RNase H (an enzyme that degrades the RNA strand of RNA-DNA hybrids) and DNA polymerase I (using its 5’ to 3’ exonuclease activity).
Step 2: After that DNA polymerase I will fill the gap between Okazaki fragments with necessary deoxyribonucleotides in 5’→3′ direction by a process called nick translation (Using its 5’ to 3’ polymerase activity).
In nick translation, DNA polymerase I recognize and bind to the DNA chain. In this way, the enzyme moves the nick along the lagging strand. After completing 10 or 12 cycles of hydrolysis and polymerization, DNA polymerase I dissociates from the DNA, leaving behind two Okazaki fragments that are separated by a nick in the phosphodiester backbone.
Step 3: Formation of a phosphodiester linkage between the 3′-hydroxyl group at the end of one Okazaki fragment and the 5′-phosphate group of an adjacent Okazaki fragment. This step is catalyzed by DNA ligase.
Refer my lecture notes on DNA ligases for more details.