Dna replication presentation

1. Genome Replication
By- Sanju Sah
St. Xavier’s College, Maitighar, Kathmandu
Department of Microbiology

2. What is Replication?
• In DNA replication two identical daughter DNA
molecules are formed from a single DNA
molecule
• This unique feature of DNA no other biological
molecule do so.

3. • DNA replication is the process of producing two
identical copies of DNA from one
original DNA molecule
• DNA is made up of a double helix of
two complementary strands
• During replication, these strands are separated and
each strand of the original DNA molecule serves as a
template for the production of its counterpart, a
process referred to as semiconservative replication.
• As a result of semi-conservative replication, each of the
new double helix will be composed of an original DNA
strand as well as a newly synthesized strand.

4. DNA Replication
• First question of discussion was whether the DNA replication
is Semiconservative or Conservative?
Semiconservative replication
• Watson and crick model
• One strand from parent DNA molecule and another is newly
formed
Conservative replication
• Both strand are in one molecule are from Parent and newly
formed DNA molecule comprising both newly synthesized
DNA strand.
Dispersive Replication
• Both strands contains the DNA from parent molecule and
newly synthesized DNA.

6. Watson and Crick Model of Replication
• During DNA replication each of two strand serve as template.
• DNA replication is semiconservative i.e. Newly formed DNA
molecule consist of one strand from parent molecule and
another strand is newly synthesized.

7. Messelson-Stahl experimetn to confirm the
Semiconservative model.
• This experiment allowed the differentiation of newly
synthesized DNA strand and parental DNA strand.
• E. coli was grown in the media containing either normal
isotope of nitrogen (14N) or heavy isotope (15N).
• DNA was extracted from both of the condition and was
performed density gradient centrifugation.
- Heavy DNA was at higher density than normal DNA.
• Bacteria grown in 15N containing media were transferred to
14N containing media.
- Newly synthesized DNA strand had 14N DNA strand while
parental strand had 15N DNA

8. Messelson-Stahl experiment contd...
• They checked the composition of resulting DNA molecule by
density gradient centrifugation.
- Found an intermediate band
- Indicating a hybrid molecule
- Containing both 14N and 15N DNA molecule

10. Semi-discontinuous model of DNA replication
• Replication of DNA occurs by forming the
complementary strand to both of DNA strand, thus
both of the DNA strand acts as the template
strand.
• Two strand of the DNA are anti-parallel to each
other, one strand runs in 5’-3’ direction where as
complementary strand runs in 3’-5’ direction.
• Replication is always in 5’-3’ direction.
• During the replication the DNA strand which runs in
3’-5’ direction is replicated in continuous manner
while the DNA strand running in 5’-3’ direction is
replicated in discontinuous manner.
• DNA strand replicated in continuous way is known
as the leading strand and DNA strand which is
replicated in discontinuous manner is known as

11. • Only one complementary strand is formed while replicating
leading strand while a number of fragments are formed while
replicating lagging strand.
• These fragments are 1000-2000 nt. long and are known as
Okazaki fragments.
• These Okazaki fragments are joined together later on by DNA
ligase to form a complete DNA strand.

12. Cell Cycle

13. Mechanism of replication
• Replication involves three step:
1)Initiation
2)Elongation
3)Termination

15. Enzymes and proteins of DNA
replication
• Nuclease
• DNA polymerase
• DNA ligase
• Primase
• Helicase
• Single strand DNA binding protein (SSBPs)
• Topoisomerase (DNA gyrase)

16. Nuclease
• A nuclease is an enzyme capable of cleaving
the phosphodiester bonds between monomers of nucleic
acids
• There are two primary classifications based on the locus of
activity.
• Exonucleases digest nucleic acids from the ends.
• Endonucleases act on regions in the middle of target
molecules.
• They are further sub divided into deoxyribonucleases
(Dnase) and ribonucleases (Rnase).
• The former acts on DNA, the latter on RNA
• Exonucleases are of two types 5l🡪3l exonuclease and 3l🡪5l
exonuclease

17. • A restriction enzyme or restriction endonuclease is an enzyme that
cleaves DNA into fragments at or near specific recognition sites within
the molecule known as restriction sites
• To cut DNA, all restriction enzymes make two incisions, once through
each sugar-phosphate backbone (i.e. each strand) of the DNA double
helix
• Restriction enzymes recognize a specific sequence of nucleotides and
produce a double-stranded cut in the DNA.
• The recognition sequences can also be classified by the number of
bases in its recognition site, usually between 4 and 8 bases

18. DNA polymerase
• DNA polymerases are enzymes that carry out synthesis
of DNA molecules using deoxyribonucleotides
• Prokaryotic DNA polymerase
• Three major DNA polymerases are known in E.coli and
includes DNA polymerase I, II and III
• DNA polymerase I is repair polymerase with 5‘🡪3' polymerase
and both 3‘🡪5' and 5‘🡪3' exonuclease activity. It is involved
in processing of Okazaki fragments generated during lagging
strand synthesis
• DNA polymerase II has 3‘🡪5' exonuclease activity and
participates in DNA repair with 5‘🡪3' polymerase activity

19. • DNA polymerase III is a multimeric holoenzyme having 10
subunits and is the primary enzyme involved in DNA replication
in E. coli. It has 3‘🡪5' exonuclease activity and 5‘🡪3' polymerase
activity
• Prokaryotic DNA is replicated by DNA polymerase III in the 5′
to 3′ direction at a rate of 1000 nucleotides per second.
• Subunits includes alpha (α), beta (β), epsilon (ε), theta (θ), tau (τ),
gamma(γ), delta (δ), delta dash (δl), chi (χ) and psi (ψ)
• Core enzyme includes alpha (α), beta (β) and theta (θ)

20. Eukaryotic DNA polymerases

21. DNA helicase
• They are motor proteins that move
directionally along a nucleic
acid phosphodiester backbone, separating
two annealed nucleic acid strands
(i.e., DNA, RNA, or RNA-DNA hybrid) and
establish replication fork using energy derived
from ATP hydrolysis, a process characterized
by the breaking of hydrogen
bonds between annealed nucleotide bases

22. DNA Topoisomerases
• DNA Topoisomerases are 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
• Class I DNA topoisomerase break one strand of
DNA helix
• Produce temporary single strand breaks in DNA
one at a time
• Class II DNA topoisomerase break both strand of
DNA helix

23. DNA primase
• DNA primase is an enzyme involved in the replication of DNA and is a
type of RNA polymerase.
• Primase catalyzes the synthesis of a short RNA (or DNA in some
organisms) segment called a primer complementary to
a ssDNA template.
• Primase is of key importance in DNA replication because no known
replicative DNA polymerases can initiate the synthesis of a DNA strand
without an initial RNA or DNA primer (for temporary DNA elongation).
• After this elongation the RNA piece is removed by a 5' to
3' exonuclease and refilled with DNA.
• In bacteria, primase binds to the DNA helicase forming a complex called
the primosome. Primase is activated by DNA helicase where it then
synthesizes a short RNA primer approximately 11 ±1 nucleotides long,
to which new nucleotides can be added by DNA polymerase.

24. DNA ligase
• DNA ligase is a specific type of enzyme, a ligase, that facilitates the
joining of DNA strands together by catalyzing the formation of
a phosphodiester bond.
• The mechanism of DNA ligase is to form
two covalent phosphodiester bonds between 3' hydroxyl ends of
one nucleotide ("acceptor"), with the 5' phosphate end of another
("donor") at expense of ATP

25. Terms used in DNA replication
• Replicon is a DNA molecule, or a region of DNA,
that replicates from a single origin of replication
• For most prokaryotic chromosomes, the replicon is
the entire chromosome
• For eukaryotic chromosomes, there are multiple
replicons per chromosome
• Replisome is a complex molecular machine that
carries out replication of DNA and includes several
enzymes and proteins such as helicase,
topoisomerase, SSB, primase, DNA polymerase,
RNAse, ligase etc

26. DNA replication in Prokaryotes
In Prokaryotes, DNA replication occurs
principally by three methods
1. Theta mode
2. Sigma mode
3. Linear mode

27. References:
Bruce Albert: Molecular biology of cell.
Lodish et al, Molecular cell Biology, 5th edition
R. Weaver, Molecular biology, 2nd edition