DNA Replication Definition

DNA replication is the process of producing two identical copies of DNA from

one original DNA molecule.
 DNA is made up of millions of nucleotides, which are composed of
deoxyribose sugar, with phosphate and a base. 
 The complementary pairing of these bases keeps the double strands
intact. So, to make two copies of one DNA, these hydrogen bonds in
between the bases should be broken to begin replication.
 DNA replication is semi-conservative, meaning that each strand in the
DNA acts as a template for the synthesis of a new complementary
strand. Semi conservative because once DNA molecule is synthesized
it has one strand from the parent and the other strand is a newly
formed strand. 
 DNA replication starts by taking one DNA molecule and giving two
daughter molecules, with each newly synthesized molecule containing
one new and one old strand. 
 DNA replication simply is the process by which a DNA makes a copy of
itself. Though easy as it may sound it’s a complex process happening
inside of our cells, and many enzymes, proteins, and metal ions should
work coherently to make this process happen.

Nucleases
 A nuclease is an enzyme that can cleave the phosphodiester bonds
present in between the nucleotides.
 On the basis where they cleave, they are characterized as Exo and
endonucleases.
 Exonucleases cleave nucleotides from their respective ends.
Corresponding to this fact, these exonucleases show activity from both
directions 5′ to 3′ and 3′ to 5′.
 Endonucleases act on the region in the middle of the targeted
nucleotide. They are also endonucleases that are selective to which
molecule they cleave and are sub-divided as DNase for DNA for
cleaving and RNase for RNA cleaving. Additionally, recently discovered
nucleases are also being used for gene editing such as Cas9 in the
CRISPR genome editing technique.
  Restrictive endonuclease or restriction enzymes are the ones that cleave
DNA into fragments at or near the specific recognition sites within the
molecule known as restriction sites.
 To cleave the DNA, restriction endonuclease makes two incisions, once
through each sugar-phosphate backbone of the DNA double helix.
These endonucleases recognize a specific sequence of nucleotides and
produce a double-stranded cut in the DNA.
 This specific sequence is usually 4 – 8 bases and is present in the
recognition site.
DNA Polymerase

 DNA polymerases are the enzyme that is responsible for adding new
nucleotides and synthesizing a new strand of DNA by taking the old
fragmented strand as a template.
 DNA Polymerases also possess exonuclease activity, that cuts incorrectly
added nucleotides, and allows the DNA replication to happen without
errors.
 DNA Polymerase is of many types and functions based on the cell they
are found in. 
 In prokaryotic cells, there are three DNA polymerases: DNA Polymerase
Ι, DNA Polymerase ΙΙ and DNA Polymerase ΙΙΙ.
 DNA polymerase Ι is a repair polymerase with 5′ to 3′ and 3′ to 5′
exonuclease activity. It is involved in the processing of Okazaki
fragments during lagging strand synthesis.
 DNA polymerase ΙΙ has 3′ to 5′ exonuclease activity and participated in
DNA repair with 5′ to 3′ polymerase activity.
 DNA polymerase ΙΙΙ is the primary enzyme involved in the DNA
replication of E.coli. It has 3′ to 5′ exonuclease activity and 5′ to 3′
polymerase activity.
 In eukaryotic cells, there are five DNA polymerases: DNA Polymerase α,
β, γ, δ and ε 
  DNA polymerase α is a repair polymerase, with 3′ to 5′ exonucleases
activities and 5′ to 3′ polymerase activities.
 DNA Polymerase β is a repair polymerase.
 DNA Polymerase γ shows polymerase activity 5′ to 3′ and exonucleases
activity 3′ to 5′, it is involved in Mitochondrial DNA replication 
 DNA Polymerase δ shows 3′ to 5′ exonuclease activity and 5′ to 3′
polymerase activity. This enzyme is involved in lagging strand
synthesis.
 DNA Polymerase ε shows 3′ to 5′ and 5′ to 3′ exonucleases activities.
This enzyme not only repairs but also synthesizes the leading strand
efficiently in a 5′ to 3′ direction. It is the prime enzyme involved in DNA
replication.
DNA Ligase

 DNA ligase is a specific type of enzyme that facilitates the joining of

DNA strands together by catalyzing the formation of a phosphodiester
bond.
 This enzyme joins the 3′ hydroxyl group of one nucleotide with the 5′
phosphate end of another nucleotide at an expense of ATP.

DNA helicase
 DNA helicase is a motor protein that moves directionally along a nucleic
acid phosphodiester backbone, separating two nucleotides of DNA
molecule.
 They separate double-stranded DNA molecules into single strands
allowing each strand to be copied.
 During DNA replication, this DNA helicase unwinds DNA at the origin, a
site where the replication is to be initiated.
  DNA helicase continues to unwind the double helix of DNA and thus
forms a structure called replication fork, named after the forked
appearance of two strands of DNA when unzipped apart. 
 It is an energy-driven process as it involves the breaking of Hydrogen
bonds between annealed nucleotide bases. 
DNA primase
 Primase is an enzyme that is capable to synthesize short stretches
of RNAsequences known as a primer.
 Primers are an integral part of DNA replication. These primers serve as
an initiating site for the addition of nucleotides by DNA polymerase. 
 DNA polymerase can only add nucleotide at pre-existing 3′ Hydroxyl
group which is thus provided by the primers.
 As we can see that primers are short stretches of RNA, but replication is
of DNA, so therefore after elongation of the chains of nucleotides,
these primers are replaced by DNA. 
DNA topoisomerase
 DNA topoisomerase is a class of enzymes that release helical tension
during transcription and replication by creating transient nicks within
the phosphate backbone on one or both strands of the DNA.
 This tension is aroused when the DNA molecule unwinds due to helicase
activity and forms a replication fork. The progress of the replication
fork generates supercoils, making it hard for other machinery involved
to access the DNA molecule.
 Class Ι DNA topoisomerase makes a single-stranded break to relax the
helix and progress the process.
 Class ΙΙ DNA topoisomerase break both the strands of DNA helix, this
class of topoisomerases is also very important during the cell cycle for
the condensation of chromosomes.
Single strand binding proteins
 The single-strand binding (SSB) protein are DNA binding proteins, that
binds to single-stranded DNA to facilitate DNA replication.
 SSB proteins prevent the hardening of strands during DNA replication. It
also protects strands from nuclease degradation and prevents the
rewinding of DNA. 
  These proteins destabilize helical duplexes so that DNA polymerase can
hold onto the DNA during DNA replication, recombination, and repair.
 It also removes unwanted secondary structures on strands for easy
access of the strands to the machinery involved in DNA replication.
 Thus, SSB proteins stabilize the single-stranded DNA structure that is
important for genomic progression.
So, in summary here are the list of 7 enzymes and proteins used in DNA
Replication:
1. Nucleases
2. DNA Polymerase
3. DNA ligase
4. DNA helicase
5. DNA primase
6. DNA topoisomerase
7. Single strand binding proteins

NoStep 2: Initiation
 One strand runs from 3′ to 5′ direction towards the replication fork and
is referred to as leading strand and the other strand runs from 5′ to 3′
away from the replication fork and is referred to as lagging strands. 
 To this exposed single-stranded DNA, SSB proteins are adhered to
prevent recoiling of DNA and to stabilize it.
 After which another enzyme DNA primase comes into action to
synthesize a short stretch of RNA primer, which provides a free 3′
hydroxyl group for DNA polymerase can now add nucleotides and
extend the new chain of nucleotides.
DNA Replication. Created with BioRender.com
Step 3: Elongation
 Now that primer is added to unzipped two single-stranded DNA, these
strands now act as a template for synthesizing new DNAs.
 The enzyme DNA polymerase synthesizes new nucleotide to match the
template and add on to the free 3′ hydroxyl group provided by the
primer in each single-stranded DNA.
 The leading strand runs from 3′ to 5′ so the addition of nucleotides by
DNA polymerase happens from 5′ to 3′ direction. As the replication
fork progresses the addition of nucleotide is continuous thus only
requiring the primer once.
 However, lagging strands is antiparallel and run from the 5′ to 3′
direction, the continuous addition of nucleotides is not possible as the
replication fork progresses, DNA polymerase cannot add
 complementary nucleotides to the 5′ end. Therefore, multiple primers
are required.
 Due to this phenomenon, the DNA nucleotides synthesis from lagging
strands occurs in fragments. These fragments are termed Okazaki
fragments.
 Hence, the leading strand using only one primer synthesizes nucleotides
continuously, while the lagging strand uses multiple primers and thus
synthesizes nucleotides discontinuously. 
Step 4: Termination
 RNA primers of both leading and lagging strands are cleaved out or
degraded by exonucleases activity of DNA polymerase, and the nicks
or gaps so formed are filled with DNA and sealed by the enzyme DNA
ligase.
 DNA polymerase also shows proofreading activity and check, remove
and replace any errors.
 Interestingly, in eukaryotic organisms having linear DNA, when RNA
primer at 5′ end of daughter strand is removed, there is not a
preceding 3′ OH such that DNA polymerase can use it to replace with
DNA.
 So, at the 5′ end of daughter strands, there is a gap (missing DNA). This
missing DNA can cause a loss of information contained in that region.
This gap must be filled before the next round of replication.
 For solving this end replication problem, researchers have found that
linear ends of DNA called telomere are used which contain specific G:
C rich repeats. These sequences are known as telomere sequences.
 These telomere sequences do not code anything but are essential to fill
in the gap in the daughter strand and maintain the integrity of DNA.
 Eventually, the replication forks terminate at terminating recognizing
sequences (ter).
 The ter sequences are of around 23 base pairs which facilitate as the
binding sites for TUS protein.
 This ter- TUS complex arrest replication fork and terminate.
So, in summary, these are the 4 steps of DNA Replication:
1. Formation of Replication Fork
2. Initiation
 3. Elongation
4. Termination
Applications of DNA Replication
 DNA replication makes the transfer of genetic information from one generation to
another possible.
 It is an important phenomenon happening inside our cells, that allows the body to
maintain homeostasis and integrity of the body.
 With the available information about DNA replication, scientific communities today have
a proper idea of genome sequencing which has now been applied in different expertise
ranging from clinical diagnosis to possible treatment of genetic diseases.
 DNA replication has made sequencing of whole human genome sequencing possible.
 Cloning of genes has also been possible by DNA replication.
 Enzymes involved in DNA replication have now been greatly studied due to their wider
applications. The recent breakthrough Cas9/ CRISPR technology where nucleases are
used to cleave the desired portion of DNA and replace it with required nucleotides is
the prime example of how we can use these enzymes and make potential advancements
in them thus broadening and exploring their uses.
 Polymerase Chain Reaction uses DNA polymerases to repeatedly replicate DNA in-vitro
and has numerous applications in diagnosis, sequencing, and recombinant DNA
technology.
 The formation of complementary DNA (cDNA) can also be considered as an example of
a wider application of the enzymes involved in DNA replication.
 There are various applications of DNA replication, we can even consider that if there is
any technique involving genes, some way or the other DNA replication is applied.