General introduction about Replication and its process...

1. Dr.K.Shoba M.Sc.,M.Phil.,Ph.D
Assistant Professor,
Department of Biochemistry,
D.K.M College for Women ( Autonomous),
Vellore, Tamil Nadu, India - 632001

2.  Introduction.
 Type of DNAs.
 Modes of DNA Replication.
 Enzymes used in DNA Replication.
 Stages of DNA Replication.
 Comparison of Eukaryotic &
Prokaryotic Replication.

3. }
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DNA is a double helical structure.
There are two polynucleotide chain coiled about one another in a spiral.
Each polynucleotide chain consists of a sequence of nucleotides linked together by
phosphodiester bonds.
The two polynucleotide strands are held together in their helical configuration by
hydrogen bonding between bases in opposing strands.
The base-pairing is specific; adenine is always paired with thymine, and
guanine is always paired with cytosine.
The two strand of a DNA double helix are complementary (not identical).
Complementarity of two strands, that makes DNA uniquely suited to store
and transmit genetic information.
The base pair in DNA are stacked 3.4 Ǻ apart with 10 base pair per turn.
The sugar-phosphate backbones of two complementary strands are antiparallel .
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5. } Replication of DNA is central to all biology.
} DNA replication is a process to produce new DNA
molecules that have the same base sequence.
} Occurs during interphase of the cell cycle
} DNA replication is semi-conservative.
◦ The parent DNA strand separates into two
◦ Each strand serves as a template for new
complementary strands.
◦ The new double helix is half original.
DNA REPLICATION

6. } DNA molecule exhibit a considerable amount of
conformational flexibility.
(It is different in different physiological
conditions.)
} DNA has 3 major forms:-
1. B – form
2. A - form
3. Z – form

8. COMPARISON
B-form
Helix sense- Right
handed
A-form
Right handed
Z-form
Left handed
per turn
Base pair _ 10 11 12
Vertical rise _ 3.4 2.56 3.7 Angstroms
per base pair
Rotation per_ +36
base pair
+33 -30 degrees
Helical diameter- 19 23 18 Angstroms

9. MODES OF DNA REPLICATION

10. } Bacteria were grown in a medium containing nitrogen 15 (N15)
for several generation.
If the medium contains no other nitrogen source, the E. coli will use
N15 and incorporate it into their DNA.
Eventually, they will only have N15 .
Once the E. coli had only N15 they were put into a growing
medium contain only N14.
N15 is heavier than N14 making new incorporation of nitrogen easy
to distinguish.
The differences were measured according to the densities of the
new strands.
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The Meselson-Stahl Experiment

11. } Molecule of different density separated by equilibrium density
gradient centrifugation.
Meselson and Stahl were able to distinguish between the three possible
mode of DNA replication by following the changes in the density of DNA
of cell grown on N15 and then transferred to N14 medium for various period
of time. So it is also called density transfer experiment.
}
} Meselson and Stahl took E. Coli and washed it to remove N15 medium. Then
transfer them to N14 medium . After one generation of growth in medium
containing N14 , had a density halfway between the densities of heavy DNA
and light DNA. This is called hybrid.
After two generation half is hybrid and half is light.
Conservative mode do not produce any hybrid.
In dispersive they should observe a shift of DNA from heavy to light.
Which was not observed.
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13. Position of Enzymes and Proteins on
DNA

14. } Primase is an enzyme that copies a DNA template strand by making a RNA
strand complementary to it.
}Primase synthesizes a short (about 10 nucleotides) RNA
primer in the 5’ to 3’ direction.
}It is product of dna G gene.
}Primase activity requires the formation of a complex of primase and at least
six proteins ( protein i , n , n’ ,n” plus the product of gene dna B and
dna C ),the complex is called the primosome.
}Helicase uses energy from the ATP to break the hydrogen bond holding the
base pair together.
}This allow the two parental strands of DNA to begin unwinding and forms two
replication fork.
}Two different helicase are involved for unwinding of each strand.
}It is coded by gene dna B.
RNA
Helicase:-
Pr im ase:-

15. single-
stranded portion of each DNA strand, preventing the
strands from reassociating and protecting
them from degradation by nucleases.
}The SSBP binds DNA as tetramer and their binding exhibit
cooperativity (i.e. the binding of one tetramer to adjacent segment of
single stranded DNA).
}SSBP binded DNA replicate over hundred times faster than
uncomplexed single stranded DNA.
}It helps to stabilize the extended single stranded template needed for
polymerization.
}It prevent folding back of DNA.
Single Stranded Binding
P}S
ri
ong
tl
ee-
is
ntr
(a
Snd
Sed
BD
PNA
) b
:i
-nding
protein (SSB) binds to the

16. } This enzyme can change the state of supercoiling in a DNA
molecules.
It relieves the tension that builds up during replication.
It catalyze the formation of negative supercoil in DNA.
It removes positive supercoils.
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DNA Polymerase I :-
DNA polymerase I is involved in removing RNA primers in the
processing of DNA after replication.
} Both DNA polymerase I and III
have the ability to "proofread" their
work by means of a 3' ◊5'
exonuclease activity.
DNA Gyrase (Type II
Topoisomerase):-

17. } DNA polymerase III is a complex enzyme containing 10 different
polypeptides (α,ε,ɣ etc.) . All those polypeptides must be present for
proper replicative function.
The 5’ to 3’ polymerase activity and the 5’to 3’ exonuclease
activity both present on the α-polypeptides of DNA polymerase III.
The 3’to 5’ proofreading activity of polymerase III present on the ε
-polypeptide.
The function of other subunit still uncertain.
It catalyse the chemical reactions for polymerization of nucleotides.
DNA polymerase III begins synthesizing DNA in the
5’◊3’direction, beginning at the 3’ end of each RNA
primer.
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DNA Polymerase
III :-

18. }
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DNA polymerase resembles a hand that grip the primer-template junction.
The speed of DNA synthesis is largely due to the processive nature of
DNA polymerase.
IN case of DNA polymerase , the degree of processivity is defined as the
average no. nucleotide added each time the enzyme binds a primer-
template junction. It can be from few nucleotides to more than 50000 bases
added per binding event.
}
Structure of DNA Polymerase
III :-

19. } DNA ligase catalyses covalent closure of the resulting single-
stranded “nick”.
} DNA ligase joins Okazaki fragments, converting them
to a
counter-helicase when it comes in contact with an advancing
helicase.
The bound Tus protein effectively halts DNA polymerase
movement.
Tus helps to end the DNA replication in prokaryotes.
continuous strand of DNA.
Terminus Utilization Substance ( TUS)
P
}
rI
ot is
tea
ip
nro
:t
-ein that binds to terminator sequences and
acts as a
}
}

21. } Initiation :-Occurs at the origin of replication.
} Elongation:- Involves the addition of new
nucleotides based on complementarity of the
template strand.
} Termination:- Occurs at a specific termination
site.

22. } Initiation- Occurs at the origin of replication.
} ORI is the region where process of replication starts.
This region is rich in A & T base pairs. It is easy to melt during the
initiation.
}

23. } At ori there is presence of specific base sequences regions .
} One is 9 base pair long regions which have four repeats and another is
13 base pair long have 3 repeats also called 13 mer region.
} 13 mers are A-T base pair rich regions.
} Dna –A proteins binds to 9 b.p. region ,which cause change in 13
mer region and it start open up.
} In 13 mer region due to presence of A-T b.p. ,it is easy to open as A-T
b.p. has two hydrogen bonds as compared to C-G b.p. as it contain three
hydrogen bond.
} After this there is attachment of helicase and SSB proteins and
other enzymes to carry on DNA replication.

24. Elongation:- Involves the addition of
new nucleotides based on complementarity
of the template strand.

25. } Occurs in a 5’→3’ direction.
◦ The 5’ end of the ‘new’ nucleotide attached to the 3’ end of the nucleotide
before it.
DNA is unwounded and unzipped by the enzyme Helicase.
Before DNA replication begins there must be RNA primer.
The RNA primer is made by adding complimentary RNA nucleotides to the lagging
DNA strand by hydrogen bonding of the bases.
RNA Primase then bonds the RNA nucleotides together.
DNA polymerase III creates links between the nucleotides. It
creates a strand that is complementary to the original strand.
In eukaryotes replication takes place at several places on one double helix at the
same time.
Once the double helix is unwound and unzipped the two parent strands become
the leading and lagging strands.
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PROCESS OF DNA
REPLICATION

27. }
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Replication is continuous.
There are no fragments.
DNA polymerase III adds nucleotides in the direction of
5’→3’.
◦ DNA polymerase only works in the direction of
5’→3’.

28. The lagging strand runs from 3’ to 5’ of template strand.
}
} 1.After RNA primer is in place DNA nucleotides are added by
DNA polymerase III.
2. Eventually, the segment of DNA will run into another RNA
primer.
3. The DNA segments are called Okazaki fragments.
4. Once Okazaki fragments are formed DNA polymerase I
replaces the RNA primer with DNA nucleotides.
5. DNA Ligase links the fragments together.
6. In the lagging strand DNA replication is discontinuous.
7. DNA is replicated in segments that become joined together.
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29. }In prokaryotes DNA replication terminates when replication fork reach specific
‘termination site’.
}The arrest of DNA replication in Escherichia coli is triggered by the encounter of
a replisome with a Tus protein-Ter DNA complex.
}A replication fork can pass through a Tus-Ter complex when traveling in one
direction but not the other, and the chromosomal Ter sites are oriented so
replication forks can enter, but not exit, the terminus region.
}The Tus-Ter complex acts by blocking the action of the replicative DnaB
helicase, but details of the mechanism are uncertain
}The two replication fork meet each other on the opposite end of the parental
circular DNA.
Termination:-

30. } It is done by exonuclease activity of DNA Pol. III . The role of exonuclease
become clear when it was determined that they have a strong preference
to degrade DNA containing mismatch base pair.
The removal of mismatched nucleotide is facilitated by the reduced ability
of DNA polymerase to add nucleotide an incorrectly base paired primer.
}
} As for processive DNA synthesis , proofreading occurs without releasing
the DNA from the polymerase.
When a mismatched base pair is present in the polymerase active site ,
the primer : template junction is destabilized , creating base pairs of
unpaired DNA.
The newly unpaired 3’end moves from the polymerase active site
to exonuclease active site.
The incorrect nucleotide is removed by the exonuclease.
The removal of mismatched base allow the primer :template junction to
reform and rebind to the polymerase active site , enabling DNA synthesis
to continue.
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32. Prokaryotes Eukaryotes
1. It occurs inside the cytoplasm.
2. There is single origin of replication.
3. DNA polymerase III carries out both
initiation and elongation.
1. DNA repair and gap filling are done
by DNA polymerase I.
2. RNA primer is removed by DNA
polymerase I.
3. Okazaki fragments are large,
1000- 2000 nucleotides long.
4. Replication is very rapid, some 2000
base pairs per second.
5. DNA gyrase is needed.
1. It occurs inside the nucleus.
2. Origin of replications are numerous.
3. Initiation is carried out by DNA
polymerase α while elongation by
DNA polymerase δ and ε.
4. The same are performed by
DNA polymerase β.
5. RNA primer is removed by DNA
polymerase β.
6. Okazaki fragments are short,
100- 200 nucleotides long.
7. Replication is slow, some 100
nucleotides per second.
8. DNA gyrase is not needed.