B-302 Molecular Biology and Biotechnology

DNA REPLICATION
• DNA replication is the process by which DNA makes a copy of itself during cell
division.
• It is semi-conservative, bidirectional and discontinuous
(Semi-conservative mechanism was demonstrated by Meselson and Stahl in 1958)

(Source: Cell and Molecular Biology- Concepts and Experiments- Gerald Karp. 7th Edition Wiley)

• DNA replication is an essential mechanism in enhancing cell growth, repair, and

reproduction of an organism.
OVERVIEW OF STRUCTURE OF DNA
• The structural model of DNA was initially proposed by James Watson and Francis
Crick.
• They found that DNA is a double-helical structure with two paired DNA strands with
complementary nucleotide sequences.
• The double-stranded DNA molecule has two parallel spiral nucleic acid chains that are
twisted into a double helix shape. The twisting gives the DNA its compactness.
• DNA is made up of millions of nucleotides that are molecules composed of
deoxyribose sugar with a phosphate group and a nucleobase that is attached to it.
• Each nucleotide is tightly base paired with a complementary nucleotide on the opposite
strand, i.e. with nucleobases Adenine (A) paired with Thymine (T) or Guanine (G)
paired with cytosine (C) – by Hydrogen Bonds (A=T and G= C)
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• Nucleotides are bound to each other in strands by phosphodiester bonds forming a

sugar-phosphate backbone. The bond is a linkage between the third carbon atom on the
deoxyribose sugar designated as the 3′ (three prime) and the fifth carbon atom of
another sugar on the next nucleotide designated as the 5′ (five prime), i.e. between
phosphate and two hydroxyl groups of adjacent nucleotides.

Source: https://en.wikipedia.org/wiki/File:Phosphodiester_Bond_Diagram.svg

Phosphodiester bond formation between nucleotides (5’ position and 3’ position)

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• DNA fits within the nucleus by being closely packed into tight coils known as
chromatins.
• The process takes place during cell division, (Interphase - S phase).
• The chromatins condense to form the chromosomes during cell division.
Before DNA replication, the chromatins loosen up giving the replication machinery access to
the DNA strands.
B-302 Molecular Biology and Biotechnology

DNA replication requires the cooperation of many proteins /enzymes

• DNA polymerase and DNA primase to catalyse nucleotide triphosphate
polymerization, i.e. add nucleotides to 3’end (5’ to 3’). Primase adds primers.
• DNA helicases and single-strand DNA-binding (SSB) proteins respectively to help
in opening up the DNA helix and prevent from breakage and renaturing
• DNA ligase and an enzyme that degrades RNA primers to seal together the
discontinuously synthesized lagging strand DNA fragments;
• DNA topoisomerases/gyrase to help to relieve helical winding and DNA tension
problems.
• DNA clamp is a protein which prevents elongating DNA polymerases from
dissociating from the DNA parent strand (beta subunits of the holoenzyme - DNA
polymerase).

Source: Molecular Biology of The Cell. Bruce Alberts et al. 6th Edition. Garland Science

• DNA-dependent DNA polymerase: It helps in the polymerization and catalyzes the

whole process of DNA replication with the support of other enzymes. DNA polymerase
is of three types:
• DNA Polymerase I: It is a DNA repair enzyme. It is involved in three activities:
• 5′-3′ polymerase activity
• 5′-3′ exonuclease activity
• 3′-5′ proof reading exonuclease activity
• It fills the gap between the lagging strand fragments
• DNA Polymerase II: It is responsible for primer extension and proofreading.
B-302 Molecular Biology and Biotechnology

• DNA Polymerase III : It is responsible for in vivo DNA replication. It has two subunits,
one each for a strand. The alpha subunit is the actual polymerase.
• Helicase: Helicase is the enzyme which unzips the DNA strands by breaking the
hydrogen bonds between them. Thus, it helps in the formation of the replication fork.
• Ligase: It is the enzyme which joins the discontinuous DNA strands by forming
phosphodiester bonds between 3’OH and 5’ PO4
• Primase: This enzyme helps in the synthesis of short RNA primers complementary to
the DNA template strand.
• Single-stranded Binding Proteins: It binds to single-stranded DNA and protects it from
forming secondary structures and thus keeping the strand unwound.

• In vivo, the DNA polymerase III holoenzyme dimer, the primosome and the DNA
helicases are believed to be physically associated in a large complex called a replisome.
• Each strand in a parental duplex DNA acts as a template for synthesis of a daughter
strand and remains base paired to the new strand, forming a daughter duplex.
• New strands are formed in the 5′ to 3′ direction.
• Replication begins at a sequence called an origin of replication. Each eukaryotic
chromosomal DNA molecule contains multiple replication origins.
• DNA replication generally occurs by a bidirectional mechanism in which a replication
fork forms at an origin and synthesis of each strand moves in opposite directions, with
both template strands being copied at the replication fork.
• At a replication fork, one daughter strand (the leading strand) is elongated continuously.
• The other daughter strand (the lagging strand) is formed as a series of discontinuous
Okazaki fragments from primers synthesized every few hundred nucleotides.
• The energy for polymerization comes from the hydrolysis of the dNTPs and the
resulting pyrophosphate.
Leading strand
• The leading strand is the strand of nascent DNA which is synthesized in the same
direction as the growing replication fork. This sort of DNA replication is continuous.
Lagging strand
• The lagging strand is the strand of nascent DNA whose direction of synthesis is
opposite to the direction of the growing replication fork. 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.
• The lagging strand is synthesized in short, separated segments. On the lagging
strand template, a primase "reads" the template DNA and initiates synthesis of a short
complementary RNA primer.
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• A DNA polymerase extends the primed segments, forming Okazaki Fragments. This
sort of DNA replication is discontinuous.
• The RNA primers are then removed and replaced with DNA, and the fragments of DNA
are joined together by DNA ligase.
• All origins contain AT-rich sequences where the strands initially separate. AT-rich
regions are more easily opened than GC-rich.

Source: https://en.wikipedia.org/wiki/File:DNA_replication_en.svg

Steps involved in DNA Replication

INITIATION
 The two strands of DNA unwind at the origin of replication.
 Helicase opens the DNA and replication fork is formed at the point of separation of
strands.
 The DNA is coated by the single-strand binding proteins around the replication fork to
prevent rewinding of DNA.
 Topoisomerase prevents the supercoiling of DNA and release the tension.
 RNA primers are synthesised by primase complementary to the DNA strand.
 Initiation in prokaryotes is at a single site OriC.
 There are multiple origin sites in eukaryotic DNA
 In E.coli. the primary initiator protein is DnaA
 In Yeast, this is the Origin Recognition Complex (ORC)
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 Sequences used by initiator proteins tend to be "AT-rich" (rich in adenine and thymine
bases), because A-T base pairs have two hydrogen bonds (rather than the three formed
in a C-G pair) and thus are easier to strand-separate.
 In eukaryotes, the origin recognition complex that is formed, catalyses the assembly of
initiator proteins into the pre-replication complex.

Source: Molecular Biology of The Cell. Bruce Alberts et al. 6th Edition. Garland Science
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ELONGATION
 DNA polymerase III starts adding nucleotides at the end of the primers.
 The leading and lagging strands continue to elongate with the formation of Okazaki
fragments in the lagging strand.

Source: Molecular Biology of The Cell. Bruce Alberts et al. 6th Edition. Garland Science
B-302 Molecular Biology and Biotechnology

TERMINATION
 The primers are removed and the gaps are filled with DNA Polymerase I and sealed by
ligase.
 Termination occurs when a termination site sequence in the DNA is reached, and a
protein binds to this sequence to physically stop DNA replication, the DNA replication
terminus site-binding protein, Ter protein, an inhibitor of DnaB helicase which works
along with a Tus factor (forming a complex).

Prokaryotic and Eukaryotic DNA Replication (differences)

• The DNA replication in eukaryotes is similar to the DNA replication in prokaryotes
with a little difference.
• All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are
circular.

Source: Molecular Biology of The Cell. Bruce Alberts et al. 6th Edition. Garland Science
B-302 Molecular Biology and Biotechnology

• Cells of higher organisms may have a thousand times as much DNA as this bacterium,
yet their polymerases incorporate nucleotides into DNA at much slower rates. To
accommodate these differences, eukaryotic cells replicate their genome in small
portions, termed replicons. Each replicon has its own origin from which replication
forks proceed outward in both directions

• The initiation process is more complex in eukaryotes than prokaryotes. The initiator
protein complex in bacteria is DnaA-ATP, Helicase is DnaB, Topoisomerase is DNA
gyrase.
• Prokaryotic chromosomes replicate as single units called replicons while in eukaryotes,
there are multiple origin of replication present.
• In contrast to prokaryotes, eukaryotic replicons can only initiate once per cell cycle.
• A pre-replication complex is made with other initiator proteins.
• Origin of replication is OriC in prokaryotes
• The process is entirely the same but the enzymes used are different, e.g. in eukaryotes,
the polymerization process is carried out by the enzyme Pol δ (delta Polymerase helped
by Polymerase alpha and epsilon), whereas in prokaryotes it is done by DNA Pol III
having two subunits (helped by Polymerase I and II)

(Source: Cell and Molecular Biology- Concepts and Experiments- Gerald Karp. 7th Edition Wiley)

Replication fork in eukaryotes

B-302 Molecular Biology and Biotechnology

Comparison of Prokaryotic and Eukaryotic Proteins in Replication

(Source: Cell and Molecular Biology- Concepts and Experiments- Gerald Karp. 7th Edition Wiley)

Regulation of DNA Replication

 Regulatory mechanisms for DNA replication are central to the control of the cell-cycle
in Eukaryotic cells.
 Replication of the eukaryotic genome occurs only once during the S-phase of each cell
cycle.
 The signal transduction pathway is the biochemical mechanism by which the growth
factor signal to grow is communicated from the outside of the cell into the nucleus to
ultimately cause that cell to begin replication and growth.
 The genes that encode elements of the signal transduction pathway are proto-
oncogenes, genes that when altered can cause cancer.
 Eukaryotic DNA replication is regulated at various stages to ensure all
chromosomes replicate once and only once per cell cycle.
 Cell cycle regulation by protein phosphorylation ensures that pre-RC assembly can
only occur in G1 phase, whereas helicase activation and loading can only occur in S
phase.
 In Prokaryotes, the replication origin and the initiator protein DnaA are the main
targets for regulation of chromosome replication (as in bacteria).
 The origin bears multiple DnaA binding sites, while DnaA contains ATP/ADP-binding
and DNA-binding domains. When enough ATP-DnaA has accumulated in the cell, an
active initiation complex can be formed at the origin resulting in strand opening and
recruitment of the replicative helicase.
 In Escherichia coli, oriC activity is directly regulated by DNA methylation and
specific oriC-binding proteins. Regulations by oriC-binding proteins are also
conserved in bacteria.
 DnaA activity is regulated by proteins that stimulate ATP-DnaA hydrolysis, yielding
inactive ADP-DnaA. Regulation of DnaA gene expression is also important for
initiation and is conserved in bacteria.
 The S phase CDK complexes stimulate the onset of organized DNA synthesis. The
machinery ensures that each chromosome is replicated only once.
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(CDK complexes are specific protein kinases made up of a regulatory subunit and a
catalytic subunit. The regulatory subunits are called cyclins and the catalytic subunits
are called cyclin-dependent kinases (CDKs)).
 A novel mammalian kinase, Cdc7-ASK (Activator of S phase Kinase), plays a key role
at the entry into S phase as a molecular switch for DNA replication. This kinase is
specially activated during S phase and triggers DNA replication by phosphorylating an
essential DNA helicase component of the replication complex.
 Certain proto-oncogenes and tumour suppressor genes regulate the passage of cells
from G1 to S phase.

Further Reading
Cell and Molecular Biology. P.K. Gupta. Rastogi Publications
Cell Biology, Genetics, Mol.Biology, Evolution and Ecology. P.S. Verma. S.Chand Publication
Molecular Biology of The Cell. Bruce Alberts et al. 6th Edition. Garland Science
Cell and Molecular Biology- Concepts and Experiments- Gerald Karp. 7th Edition Wiley