The following points highlight the three main phases of DNA replication in prokaryotes. The phases are: 1. Initiation 2. Elongation 3. Termination.
Phase # 1. Replication Initiation:
Replication initiation involves the following events:
(1) Recognition of origin,
(2) DNA melting, i.e., separation of the two strands in the origin region,
(3) Stabilization of the single strands,
(4) Assembly of primosome at the two forks so produced, and finally, and
(5) Start of synthesis of the two daughter strands.
Replication initiation in E. coli requires 6 proteins, viz., DnaA, DnaB, DnaC, HU, gyrase and SSBP (single strand binding proteins). First, 2-4 molecules of DnaA bind oriC; this results in the folding of the origin Oric DNA around DnaA aggregate. As a result, DnaA now induces melting at OricC. Now an aggregate having 6 molecules each of DnaB and DnaC binds to each of the three separate single-stranded regions produced by DnaA.
The aggregate eventually displaces DnaA, and DnaC loads the DnaB hexamer at the two forks produced by melting. DnaB functions as helicase and begins to unwind the DNA. Gyrase facilitates unwinding by helicase as it provides a swivel. SSBP bind to the single-stranded regions so produced and stabilize them. Initiation of replication generally requires ~ 60 bp of unwound DNA, and the process consumes ATP. One DnaB hexamer binds to each of the two forks produced by unwinding at the origin (Fig. 28.10).
Once a replication fork is generated, primosome assembles at the origin, and initiates primer synthesis; this is called priming. Priming occurs only once and at the origin for the replication of the leading strand. But for replication of the lagging strand, priming occurs repeatedly at intervals of 1000 to 2000 bases.
Priming reaction at oriC is rather simple the primosome consists of a single protein, DnaG. DnaG needs to be activated by DnaB. DnaB also serves as helicase, while DnaG carries out primer synthesis; primers of 15-50 bases are normally synthesized.
The replication fork proceeds in the 5’→ 3′ direction in relation to the lagging strand. The replication fork advances and generates a single-stranded region of the lagging strand bound to SSBP ahead of the primosome. The primosome moves along this single-stranded region. When the primosome reaches a site at which priming can occur, it synthesizes an RNA primer. This primer sponsors synthesis of a new Okazaki fragment (Fig. 28.11).
Energy from ATP is required during:
(1) Melting of DNA by DnaA,
(2) Release of DnaB at the forks by DnaC,
(3) Helicase action of DnaB,
(4) Swivel action of DNA gyrase,
(5) Activation of primase DnaG by DnaB, and
(6) Activation of DNA polymerase III to begin replication.
Phase # 2. Primer Elangation (DNA Replication):
Once the primer has been synthesized, DNA synthesis is taken up by replisome, which is a complex of proteins. In E. coli, DNA replication activity is provided by DNA polymerase III component of replisome.
Each E. coli cell has ~ 10 molecules of DNA polymerase III; most of these molecules are associated with replication forks. The complete enzyme, holoenzyme, molecule has the following subunits; α2, θ2, ԑ2 ϒ, χ, ψ, δ, δ’, τ 2, β4 (Fig. 28.12).
The enzyme is assembled at the replication fork as follows:
1. First, the γ- δ complex (subunits γ δ δ’ Χ ψ) or ‘clamp loader’ and a pair of β subunit (the ‘clamp’) recognize the primed-template and bind to it.
2. They now attach to a catalytic core (∝ θ ԑ subunits).
3. Subunit τ now joins the complex. It brings two more β subunits and another catalytic core to the complex. This generates a DNA polymerase III holoenzyme.
According to one model, a single holo-enzyme molecule functions at one replication fork. Each holoenzyme molecule has 2 catalytic cores; one catalytic core catalyzes the replication of leading strand, while the other catalyzes that of the lagging strand (Fig. 28.13).
In the case of leading strand, the catalytic core extends the primer one nucleotide at a time. DnaB progressively unwinds the duplex and the replication fork moves along.
Replication of the lagging strand will begin sometime later. When DnaB associated with the advancing fork reaches a site suitable for priming, it activates DnaG to synthesize a primer in the normal 5′ → 3′ direction, i.e., moving from the fork toward the origin. When the primer become 10-14 bases long, the other catalytic core begins to elongate this primer in the 5’→ 3′ direction.
The lagging strand, is in effect, pulled up by the replisome in the process of replication; it therefore, forms a progressively larger loop between the fork and the replisome (Fig. 28.13).
When the replisome reaches the 5′-end of the primer of the previous Okazaki fragment, it stops replication and dissociates from the lagging strand. Meanwhile DNAB continues to move forward with the replication fork. When it reaches the appropriate site, it again induces primer synthesis by DnaG and the events described above take place again.
In eukaryotes, two different enzymes are used to replicate the leading and the lagging strands. Leading strand is replicated by DNA polymerase δ, while replication of the lagging strand is due to DNA polymerase ԑ. Primase activity is due to DNA polymerase α, which primes both the leading and the lagging strands. It also begins to synthesize DNA using this primer, but is soon replaced by DNA polymerase δ (in the case of leading strand) and ԑ (in the case of lagging strand).
In E. coli, termination is signalled by specific sequences called ter elements, which serve as a binding site for protein Tus. Tus protein binds to ter element and stops DnaB from unwinding DNA.
This stops the movement of the replication fork. The leading strand is replicated up to the ter element, while the lagging strand replication is stopped 50-100 bp before the ter element. It is significant that Tus protein is able to stop fork movement in only one direction.