DNA Replication
Abdur Rehman
PhD Biochemistry
Replication Facts
• DNA must be copied before a cell divides
• DNA is copied during the S or synthesis phase of interphase
• New cells will need identical DNA strands
• Interphase → G1, S and G2 Phase
• Mitosis → Prophase, Metaphase,Anaphase,Telophase
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Synthesis Phase (S phase)
• S phase during interphase of the cell
cycle
• Nucleus of eukaryotes S
DNA replication takes phase
place in the S phase.
G1 Interphase G2
Mitosis
-prophase
-metaphase
-anaphase
-telophase
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Steps in prokaryotic DNA
synthesis
• Two strands of the DNA double helix are
separated,
• Each can serve as a template for the
replication of a new complementary strand.
• This produces two daughter molecules,
each of which contains two DNA strands
with an antiparallel orientation
• This process is called semiconservative
replication
Steps in prokaryotic DNA synthesis
Separation of the two complementary DNA strands
Formation of the replication fork
Direction of DNA replication
RNA primer
Chain elongation
Excision of RNA primers and their replacement by DNA
DNA ligase
Termination
Separation of the two complementary DNA
strands
• DNA replication begins at a single, unique nucleotide sequence, a site
called the origin of replication, or ori
• The ori includes short, AT-rich segments that facilitate melting
• In Prokaryotes → Single ori
• In eukaryotes → Multiple ori
Formation of the replication fork
• As the two strands unwind and separate, synthesis occurs at two
replication forks that move away from the origin in opposite directions
(bidirectionally) generating a replication bubble
• A group of proteins are required to initiate the replication and to
recognize the ori which are responsible for:
i. maintaining the separation of the parental strands
ii. unwinding the double helix ahead of the advancing replication fork
Proteins required to form pre-priming complex
I. DnaA Protein
→binds to specific nucleotide sequences within the ori
→Melt AT-rich regions in the ori
→Melting is adenosine triphosphate (ATP) dependent
II. DNA Helicase (dnaB protein)
• Actually an enzyme → unwind the DNA double helix
• Helicases require energy provided by ATP
• Unwinding at the replication fork causes supercoiling in other regions
of the DNA molecule
Proteins required to form pre-priming complex
III. Single strand binding proteins
• This protein binds to the ssDNA generated by helicases
• Binding is cooperative
• The SSB proteins are not enzymes
• These proteins keep the two strands of DNA separated in the area of the
replication origin,
• providing the single-stranded template required by polymerases,
• protect the DNA from nucleases that degrade ssDNA.
DNA topoisomerases
• DNA strands separation leads
to formation of Positive
supercoils in the region of DNA
ahead of the replication fork as
a result of over-winding
• DNA Topoisomerases are
responsible for removing
supercoils in the helix by
transiently cleaving one or both
DNA strands.
DNA topoisomerases Cont..
▪ Type I DNA Topoisomerases
• Cut one strand of DNA
• Does not require ATP
▪ Type II DNA Topoisomerases
• Cut both strands
• Require ATP
• DNA gyrase, a type II topoisomerase found in
bacteria and plants, has the unusual property of
being able to introduce negative supercoils into
circular DNA
• Negative supercoils neutralize the positive
supercoils introduced during opening of the
double helix
• Anticancer agents, such as the camptothecins,
target human type I topoisomerases,
• Etoposide targets human type II topoisomerases.
• fluoroquinolones targets Bacterial DNA gyrase
and act as antimicrobial agents
DNA polymerases
• DNA polymerases: Chief enzyme mainly involved in repair
and deoxynucleotide polymerization.
• In prokaryotes: Three types of DNA polymerases found.
– DNA polymerase I,
– DNA polymerase II and
– DNA polymerase III.
• In eukaryotes: There are five types of DNA polymerases
– α, ε, β, γ and δ.
DNA Polymerases
Direction of DNA replication
• The DNA polymerases for DNA Replication
• Read” the parental nucleotide sequences in the 3ʹ→5ʹ direction,
• Synthesize the new DNA strands only in the 5ʹ→3ʹ (antiparallel) direction.
• two newly synthesized DNA strands must grow in opposite directions,
• One in the 5ʹ→3ʹ direction toward the replication fork and one in the 5ʹ→3ʹ
direction away from the replication fork
Leading and Lagging strands
1. Leading strand:
• The DNA is synthesized continuously in 5’ → 3’ direction with same over-all
forward direction.
• The strand that is being copied in the direction of the advancing replication
fork is called the leading strand and is synthesized continuously.
2. Lagging strand (Retrograde strand):
• The DNA is synthesized in a discontinuous manner.
• The strand that is being copied in the direction away from the replication fork
• Okazaki fragments
• Okazaki fragments, are eventually joined (ligated) to become a single,
continuous strand
RNA primer
• DNA polymerases cannot initiate synthesis of a complementary strand of
DNA on a totally single-stranded template.
• They require an RNA primer, which is a short, double-stranded region
consisting of RNA base paired to the DNA template, with a free
hydroxyl group on the 3ʹ-end of the RNA strand
• This hydroxyl group serves as the first acceptor of a deoxynucleotide
by action of a DNA polymerase
Primase (DnaG)
• A specific RNA polymerase, called primase (DnaG), synthesizes the short
stretches of RNA (approximately ten nucleotides long)
• Short RNA sequences are constantly being synthesized at the
replication fork on the lagging strand,
• Only one RNA sequence at the origin of replication is required on the
leading strand
Chain elongation
• DNA polymerases (DNA pol) elongate a new DNA strand by adding
deoxyribonucleotides, one at a time, to the 3ʹ-end of the growing chain
• DNA polymerase III:
• Multisubunit enzyme,
• Adds new nucleotides to the 3ʹ- hydroxyl group of the RNA primer
• DNA pol III is a highly “processive” enzyme (that is, it remains bound to
the template strand as it moves along and does not diffuse away and
then rebind before adding each new nucleotide
Chain elongation
Cont..
• All four substrates
– Deoxyadenosine triphosphate [dATP],
– Deoxythymidine triphosphate [dTTP],
– Deoxycytidine triphosphate [dCTP], and
– Deoxyguanosine triphosphate [dGTP])
• Must be present for DNA elongation to occur
– If one of the four is in short supply, DNA synthesis stops
when that nucleotide is depleted
Proofreading of newly synthesized DNA
• Misreading of the template sequence could result in deleterious, perhaps
lethal, mutations
• To ensure replication fidelity, DNA pol III has a “proofreading” activity
(3ʹ→5ʹ exonuclease) in addition to its 5ʹ→3ʹ polymerase activity
• DNA pol III checks every newly added nucleotide,
• correctly matched to its complementary base on the template.
• If it is not, the 3ʹ→5ʹ exonuclease activity removes the error
DNA ligase
• The final phosphodiester linkage between the 5ʹ-phosphate group on
the DNA chain synthesized by DNA pol III and the 3ʹ-hydroxyl group on
the chain made by DNA pol I is catalyzed by DNA ligase
• ATP is required
Termination
• Replication termination in E. coli is mediated by sequence-specific
binding of the protein,
• Tus (terminus utilization substance) to replication termination sites (ter
sites) on the DNA, stopping the movement of DNA polymerase
References
• Lippincott’s Illustrated Reviews:BiochemistryFifth
Edition