Key points in DNA Replication:

• DNA replication is semiconservative. Each strand in the double helix acts as a template for
synthesis of a new, complementary strand.
• New DNA is made by enzymes called DNA polymerases, which require a template and a
primer (starter) and synthesize DNA in the 5' to 3' direction.
• During DNA replication, one new strand (the leading strand) is made as a continuous piece.
The other (the lagging strand) is made in small pieces.
• DNA replication requires other enzymes in addition to DNA polymerase, including DNA
primase, DNA helicase, DNA ligase, and topoisomerase.

Prokaryotic DNA Replication

DNA replication is fundamental process occurring in all living organism to copy their DNA.

The process is called replication in sense that each strand of double strand DNA serve as

template for reproduction of complementary strand.

DNA replication employs a large number of proteins and enzymes, each of which plays a

critical role during the process. One of the key players is the enzyme DNA polymerase, which

adds nucleotides one by one to the growing DNA chain that are complementary to the template

strand. The addition of nucleotides requires energy; this energy is obtained from the nucleotides

that have three phosphates attached to them, similar to ATP which has three phosphate groups

attached. When the bond between the phosphates is broken, the energy released is used to form

the phosphodiester bond between the incoming nucleotide and the growing chain.

Enzymes involved in Prokaryotic DNA replication

The following enzymes plays the pivotal role in the process of prokaryotic DNA replication

1. DNA polymerases
2. DNA Helicase (DnaB)
3. DNA Topoisomerase (Gyrase)
4. DNA Primase (DnaG)
1. DNA Polymerases:

DNA polymerases are the most important enzymes used in the process of DNA replication and

helps in polymerization process. It monitors the accurate incorporation of nucleotides during

the elongation process. In prokaryotes, three main types of DNA polymerases are known:

DNA polymerase I, DNA polymerase II, and DNA polymerase III.

DNA polymerase I is also known as Kohnberg enzyme. It has two subunits that helps in 5’-3’

polymerase activity and also show 3’-5’ exonuclease activity. Its main function is to remove

the RNA primers and replace it with complementary DNA sequence.

DNA polymerase II is not directly involved in the process of DNA replication, but sometimes

show 3’-5’ exonuclease activity.

DNA polymerase III is the main polymerizing enzyme which helps in the process of DNA

replication.

2. DNA Ligase:

Ligase is the enzyme which helps in sealing the gaps between Okazaki fragments of the lagging

strands.

3. Helicase (DnaB):

Helicase performs unwinding of the double strand of DNA by dissociating/breaking the

hydrogen bonds between nucleotide pairs.

4. DNA Topoisomerase (DNA Gyrase)

The activity of helicase causes the topological stress to the unwinded strand forming

supercoiled DNA. This stress is relieved by the DNA topoisomerase (DNA gyrase) by negative

supercoiling.
Mechanism of DNA replication

The whole process of DNA replication occurs in following three steps such as initiation,

elongation and termination.

A. Initiation

In E coli, the replication starts at the origin of replication or OriC which is around of 245 base

pair in length and contains DNA sequences that are highly conserved among bacterial

replication origin. Two types of conserved sequences are found at OriC, three repeats of 13 bp

called 13 mers (5’-GATCTATTTATTT-3’) and five repeats of 9 bp called 9 mers (5’-

TTATCCACA-3’).

The primosome required for primer synthesis consists of seven proteins: DnaG (primase),

DnaB (helicase), DnaC (helicase assistant), DnaT, PriA, Pri B, and PriC. At each replication

fork, the primosome is utilized once on the leading strand of DNA and repeatedly, initiating

each Okazaki fragment, on the lagging DNA strand. Initially the complex formed by PriA,

PriB, and PriC binds to DNA. Then the DnaB-DnaC helicase complex attaches along with

DnaT. This structure is referred to as the pre-primosome. Finally, DnaG will bind to the pre-

primosome forming a complete primosome.

OriC contains 11 5’-GATC-3’ repeats that are methylated on adenine (by DAM methylase) on

both strands. Only fully methylated origins can initiate replication. Immediately after DNA

synthesis, the daughter strand remains unmethylated for a short time. Hemi-methylated

daughter origins can’t be used again until they have been restored to the fully methylated state.

Initially about 20 molecules of DnaA proteins binds with 9 mer repeats along with ATP which

causes DNA to wraps around dnaA protein forming initial closed complex. The dnaA protein

and ATP trigger the opening of 13 mer repeats forming open complex.
Then two copies of dnaB proteins (helicase) binds to 13 mer repeats. This binding is facilitated

by another molecule called dnaC (helicase assistant/loader). Then this dnaB-dnaC interaction

causes dnaB ring to open which binds with each of the DNA strand. The hydrolysis of bound

ATP release dnaC leaving the dnaB bound to the DNA strand.

The binding of helicase is key step in replication initiation. dnaB migrates along the single

stranded DNA in 5’-3’ direction causing unwinding of the DNA. The activity of helicase causes

the topological stress to the unwinded strand forming supercoiled DNA. This stress is relieved

by the DNA topoisomerase (DNA gyrase) by negative supercoiling.

Simultaneously, single stranded binding protein (SSBs) binds to the separated strand and

prevents reannealing of separated strand and stabilize the strand.

The DNA polymerase cannot initiate DNA replication. So, at first primase synthesize around

10 nucleotide (RNA) along the 5’-3’ direction. Primer is closely associated with dnaB helicase

so that it is positioned to make RNA primer as ssDNA of lagging strand.

B. Elongation
Leading strand synthesis: Leading strand synthesis is more a straight forward process which

begins with the synthesis of RNA primer by primase at replication origin. DNA polymerase III

then adds the nucleotides at 3’end. The leading strand synthesis then proceed continuously

keeping pace with unwinding of replication fork until it encounter the termination sequences.

Lagging strand synthesis: The lagging strand synthesized in short fragments called Okazaki

fragments. At first RNA primer is synthesized by primase and as in leading strand DNA

polymerase III binds to RNA primer and adds dNTPS. On this level the synthesis of each

Okazaki fragments seems straight forward but the reality is quite complex.

The complexity lies in the co-ordination of leading and lagging strand synthesis. Both the

strand are synthesized by a single DNA polymerase III dimer which accomplished the looping

of template DNA of lagging strand synthesizing Okazaki fragments.

Helicase (dnaB) and primase (dnaG) constitute a functional unit within replication complex

called primosome. DNA pol III use one set of core sub unit (Core polymerase) to synthesize

leading strand and other set of core sub unit to synthesize lagging strand. In elongation steps,

helicase in front of primase and pol III, unwind the DNA at the replication fork and travel along

lagging strand template along 5’-3’ direction. DnaG primase occasionally associated with dnaB

helicase synthesizes short RNA primer. A new B-sliding clamp is then positioned at the primer

by Beta-clamp loading complex of DNA pol III.

When the Okazaki fragments synthesis is completed, the replication halted and the core sub

unit dissociates from their sliding clamps and associates with new clamp. This initiates the

synthesis of new Okazaki fragments. Both leading and lagging strand are synthesized co-

ordinately and simultaneously by a complex protein moving in 5’-3’ direction. In this way both

leading and lagging strand can be replicated at same time by a complex protein that move in

same direction.
Lagging strand synthesis is not completes until the RNA primer has been removed and the gap

between adjacent Okazaki fragments are sealed. The RNA primer are removed by exonuclease

activity (5’-3’) of DNA pol-I and replaced by DNA sequences. The gap is then sealed by DNA

ligase.

Termination

Ultimately, the two replication fork of circular E. coli chromosome meet at termination

recognizing sequences (ter). To this Ter sequence, TUS protein binds. The Ter sequence of

23 bp are arranged in such as way on the chromosome to create a trap so that the replication

fork can enter but cannot leave. Therefore, the Ter sequences function as binding site for TUS

protein.

Eukaryotic DNA replication

DNA replication in eukaryotes occur only in S-phase of cell cycle. However pre-initiation

occur in G1 phase. Due to sheer size of chromosome in eukaryotes, chromosome contains

multiple origin of replication. ARS (autonomously replicating sequence) in case of yeast is

origin for replication.

Steps in DNA Eukaryotic replication

Initiation

The first steps is the formation of pre-initiation replication complex (pre-RC). It occur in

early G1 phase and involves binding of ORC (origin recognition complex).

and remains bounded even after DNA replication occurs. Furthermore ORC is analogue of

prokaryotic dnaA protein. After binding of ORC to origin, cdc6 and cdt-1co-ordinate the

loading of MCMs (mini chromosome maintenance protein complex) to origin. MCMs complex

is thought to be major eukaryotic helicase. After binding of MCMs complex to pre-RC, cdt-l

get displaced. Then CDK & DDK phosphorylates MCMs, which activates its helicase activity.

Again DDK and CDK recruit another protein called cdc45 which then recruit all the DNA

replicating protein such that the origin get fired and replication begins.

Elongation:

The leading and lagging strands are synthesized in the similar fashion as in prokaryotic DNA

replication. In eukaryotes, once the initiation complex is formed and the cells pass into the S

phase, the complex then becomes a replisome. The eukaryotic replisome complex is

responsible for coordinating DNA replication. Replication on the leading and lagging strands

is performed by DNA polymerase ε and DNA polymerase δ. Basically proliferating cell

action. Another protein called replication factor C (RFC) act as clamp loader in eukaryotic

DNA replication.

MCMs (Helicase)

Leading strand synthesis: Leading strand synthesis is more a straight forward process which

begins with the synthesis of RNA primer by primase at replication origin. DNA polymerase ε

then adds the nucleotides at 3’end in the process of leading strand DNA synthesis. The leading

strand synthesis then proceed continuously keeping pace with unwinding of replication fork

until it encounter the termination sequences.

Lagging strand synthesis: The lagging strand synthesized in short fragments called Okazaki

fragments. At first RNA primer is synthesized by primase and as in leading strand DNA

polymerase δ binds to RNA primer and adds dNTPS for lagging strand DNA synthesis. On

this level the synthesis of each Okazaki fragments seems straight forward but the reality is quite

complex.
The complexity lies in the co-ordination of leading and lagging strand synthesis. Both the

strand are synthesized by a single DNA polymerase ε-δ dimer which accomplished the looping

of template DNA of lagging strand synthesizing Okazaki fragments.

Helicase and DNA polymerase α (Primase) constitute a functional unit within replication

complex called primosome. In elongation steps, helicase in front of primase and polymerase

εδ, unwind the DNA at the replication fork and travel along lagging strand template along 5’-

3’ direction. In eukaryotes, these single-stranded binding proteins are a heterotrimeric complex

known as replication protein A (RPA).

When the Okazaki fragments synthesis is completed, the replication halted and the core sub

unit dissociates from their sliding clamps and associates with new clamp. This initiates the

synthesis of new Okazaki fragments. Both leading and lagging strand are synthesized co-

ordinately and simultaneously by a complex protein moving in 5’-3’ direction. In this way both

leading and lagging strand can be replicated at same time by a complex protein that move in

same direction. Lagging strand synthesis is not completes until the RNA primer has been

removed and the gap between adjacent Okazaki fragments are sealed by DNA ligase. At the

end of DNA replication the RNA primer are replaced by DNA by 5’-3’exonuclease and

polymerase activity of DNA polymerase ε.

Termination

Termination of eukaryotic DNA replication requires different processes depending on whether

the chromosomes are circular or linear. Unlike linear molecules, circular chromosomes are able

to replicate the entire molecule. However, the two DNA molecules will remain linked together.

This issue is handled by decatenation of the two DNA molecules by a type II topoisomerase.

However, in eukaryotic linear DNA replication, as RNA primer is required for initiation of

DNA synthesis, the lagging strand is at a disadvantage in replicating the entire chromosome.
While the leading strand can use a single RNA primer to extend the 5' terminus of the

replicating DNA strand, multiple RNA primers are responsible for lagging strand synthesis,

creating Okazaki fragments. This leads to an issue due to the fact that DNA polymerase is only

able to add to the 3' end of the DNA strand.

The 3'-5' action of DNA polymerase along the parent strand leaves a short single-stranded DNA

(ssDNA) region at the 3' end of the parent strand when the Okazaki fragments have been

repaired. Since replication occurs in opposite directions at opposite ends of parent

chromosomes, each strand is a lagging strand at one end. Over time this would result in

progressive shortening of both daughter chromosomes. This is known as the end replication

problem. The end replication problem is handled in eukaryotic cells by telomere regions and

telomerase.

In eukaryotes, the linear end of DNA called telomere has G:C rich repeats. These sequence is

known as telomere sequence. These repeats of telomere sequence is different among different

organisms. Telomere in human cell consists of repeats of TTAGGG/AATCCC. Each species

has its own species specific telomere repeats. These telomere sequence do not codes anything

but it is essential to fill the gap in daughter strand and maintain the integrity of DNA.

There is an enzyme found in eukaryotic cell called telomerase. Telomerase is a DNA

polymerase (RNA dependent DNA polymerase) which adds many copies of telomere sequence

at 3’-OH end of template strand. Like other DNA polymerase, telomerase also adds

deoxyribonucleotide at 3’-OH end. Unlike other DNA polymerase, telomerase adds DNA at

3’-OH end of parent strand not at the daughter strand and also it synthesizes the same

sequences over and over in absence of template strand.

Thus, the parent strand become more longer than daughter strand. Now RNA polymerase

(PRIMASE) synthesize RNA primer by copying the parent strand in 5’-3’ direction using
telomere sequence as template. The DNA polymerase can now extend the primer in 5’-3’

direction by adding deoxyribonucleotide to 3’ end. The primer is now removed and it won’t be

replaced because it is an extra sequence added by copying telomere sequence. Finally the

integrity of daughter strand is maintained.