INTRODUCTION,IMPORTANCE,MODELS OF REPLICATION,STEPS INVOLVED IN REPLICATION AND REPAIR MECHANISMS

1. BIOCHEMISTRY AND GENETICS 1
TOPIC : DNA REPLICATION
BY: NOHA ARSHAD

2. Deoxyribonucleic acid (DNA)
 Deoxyribonucleic acid is a molecule composed of two chains that coil around each
other to form a double helix carrying genetic instructions for the development,
functioning, growth and reproduction of all known organisms and many viruses.

3. DNA REPLICATION
 In molecular biology, DNA replication is the biological process of producing two
identical replicas of DNA from one original DNA molecule. DNA replication occurs
in all living organisms acting as the basis for biological inheritance.
 LOCATION :
 occurs in the cytoplasm of prokaryotes
 in the nucleus of eukaryotes.

4. IMPORTANCE OF
REPLICATION:

5. IMPORTANCE :
 DNA replication is important since it creates a next copy of DNA that have to go
into one of the two daughter cells when a cell divides.
 Without replication, each cell lacks adequate hereditary fabric to give instructions
for creating proteins vital for bodily purpose
 This procedure occurs in all livelihood organisms and is the foundation for
biological legacy.
 Extreme accuracy of dna replication is necessary in order to preserve the integrity
of genome in generation.
 It is essential for continuation of life.

6. Providing variety of order of bases responsible for
each organism.
 DNA replication is important because without it the
new cells that are produce during mitosis and
meiosis would eventually die.
It is important for growth and development.
It is responsible for sequence of codons.

9. DNA REPLICATION
 Basis for inheritance
 It is semi-discontinuous
leading and lagging strands
LEADING STRAND
 Continuous synthesis
LAGGING STRAND
 Discontinuous synthesis
 okazaki fragments
 joined by ligases
 Primer is needed
RNA PRIMER
 Synthesized by primase
 Serve as starter sequence for DNA polymerase III
 It has a free 3’OH group to which first nucleotide is bound

10. COMPONENTS OF REPLICATION
 Helicase (unwinds the DNA double helix)
 Primase (lays down RNA primers)
 DNA polymerase III (main DNA synthesis enzyme)
 DNA polymerase I (replaces RNA primers with DNA)
 Ligase (fills in the gaps)

11. DNA REPLICATION :STEPS
Identification of origins of replication
Unwinding of ds DNA
Formation of replication fork
Initiation of DNA synthesis and its elongation
Termination

12. ORIGINS OF REPLICATION
The DNA REPLICATION begins at one or more sites
on DNA molecule , where there is specific sequence
of nucleotides. These sites are called origins .

13. UNWINDING OF DNA
 DNA Helicase is responsible for the unwinding of dsDNA

14. FORMATION OF REPLICATION FORK
 It is created when DNA helicase unwinds the double helix structure of the DNA.

15. FORMATION OF REPLICATION BUBBLES
 REPLICATION occurs in both directions along the length of
DNA and both strands (leading and lagging strands ) are
simultaneously replicated.
 This replication process generates replication bubbles.

16. DNA Polymerase
The DNA polymerase III and other enzymes begin a
complex process that catalyzes the addition of
nucleotides to the growing complementary strands
of DNA.
There are three DNA polymerases
o DNA Polymerase I (plays supporting role)
o DNA Polymerase II
o DNA Polymerase III (Main enzyme)

17. INITIATION:
 DNA polymerase can not initiate synthesis on its own .
 Primase constructs RNA primer complementary to parent strand.
 The RNA primers are than replaced by DNA nucleotides.
 One of the main features of DNA polymerase is that it can add
nucleotides only to a chain of nucleotides which are already paired with
parent stand.
 Another feature is that it can add nucleotides only to 3’ end of the DNA
strand.
 Replication proceeds in 5’ 3’ direction.

18. ELONGATION :
As the two strands are anti-parallel so the two strands are elongated by
different mechanism:
LEADING STRAND
 Elongates towards the replication
fork
 Built up simply by adding
nucleotides continuously to 3’ end
LAGGING STRAND
 Elongates away from the replication
fork
 Synthesized discontinuously as a
series of short segments that are
later connected
 These segments are called okazaki
fragments

19. OKAZAKI FRAGMENTS
short sequences of DNA nucleotides
approximately 100 to 200 nucleotides long in eukaryotes
 which are synthesized discontinuously
 linked together by the enzyme DNA ligase to create the
lagging strand during DNA replication.

20. ELONGATION continued……..
The DNA is further unwound and new RNA primers
are constructed
DNA Polymerase jumps 1000-2000 nucleotides
toward the replication fork to begin constructing
another okazaki fragment

22. TERMINATION:
 The last step of DNA Replication is the Termination. This process happens when
the DNA Polymerase reaches to an end of the strands.
 Each new double helix consist of one new and one old chain.
 this proves that DNA replication is based on semi conservative model.

23. TERMINATION………………………..
 Termination requires that the progress of the DNA replication fork must
stop or be blocked.
 Termination at a specific locus, when it occurs, involves the interaction
between two components:
(1) a termination site sequence in the DNA
(2) a protein which binds to this sequence to physically stop DNA
replication

24. ERRORS OF
REPLICATION

25. Error of DNA replication:
 James Watson and Francis crick published their model of the double-helix
structure of DNA in 1953.
 most replication errors were caused by what are called tautomeric shifts.
 Both the purine and pyrimidine bases in DNA exist in different chemical forms, or
tautomer, in which the protons occupy different positions in the molecule
 The Watson-Crick model required that the nucleotide bases be in their more
common "keto" form . Scientists believed that if and when a nucleotide base
shifted into its rarer tautomeric form (the "imino" or "enol" form), it result would be
base-pair mismatching.

28. Tautomeric shifts in nucleotide bases;
 The purine and pyrimidine bases in DNA exist in two different tautomers, or
chemical forms. (A) Nucleotide bases shift from their common “keto” form to their
rarer, tautomeric “enol” form. (B) In common base pair arrangements, the common
form of thymine (T) binds with the common form of adenine (A), and the common
form of cytosine (C) binds with the common form of guanine (G). (C) Rare base-
pairing arrangements result when one nucleotide in a base pair is the rare form
instead of the common form. Here, the rare form of cytosine binds to the common
form of adenine instead of guanine. The rare form of guanine binds to the common
form of thymine instead of cytosine.

29. Mutations:
 A mutation is a change that occurs in our DNA sequence, either due to mistakes
when the DNA is copied or as the result of environmental factors such as UV light
and cigarette smoke. Mutations can occur during DNA replication if errors are
made and not corrected in time.
 Incorrectly paired nucleotides that still remain following mismatch repair become
permanent mutations after the next cell division. This is because once such
mistakes are established, the cell no longer recognizes them as errors. Consider the
case of wobble-induced replication errors. When these mistakes are not corrected,
the incorrectly sequenced DNA strand serves as a template for future replication
events, causing all the base-pairings thereafter to be wrong

31. EXAMPLES:
 Sickle cell disease :named due to its characteristics sickling effect on red blood
cells, manifests via blood clots ,anemia and bouts of pain known as sickle –cell
crises while many of these symptoms can be treated with medications ,they still
significantly lower the quality of life of their carriers .

32. CORRECTION OF ERRORS
 Repair mechanisms can correct the mistakes, but in rare cases mistakes are not
corrected, leading to mutations.
 Most of the mistakes during DNA replication are promptly corrected by DNA
polymerase which proofreads the base that has just been added.
 DNA pol reads the newly-added base before adding the next one so a correction
can be made.
 It checks whether the newly-added base has paired correctly with the base in the
template strand
 If an incorrect base has been added, the enzyme makes a cut at the
phosphodiester bond and releases the incorrect nucleotide.

33. Cont.……………………….
 This is performed by the exonuclease action of DNA pol III.
 Once the incorrect nucleotide has been removed, a new one will be added again.

34. MISMATCH REPAIR :
 Some errors are not corrected during replication, but are corrected after
replication is completed;
 this type of repair is known as mismatch repair.
 The enzymes recognize the incorrectly-added nucleotide and excise it; this is then
replaced by the correct base.
 If this remains uncorrected, it may lead to more permanent damage.
 How do mismatch repair enzymes recognize which of the two bases is the
incorrect one?

35. CONT……………………
 In E. coli, after replication, the nitrogenous base adenine acquires a methyl group;
 the parental DNA strand will have methyl groups,
 whereas the newly-synthesized strand lacks them.
 DNA polymerase is able to remove the incorrectly-incorporated bases from the
newly-synthesized, non-methylated strand.

37. ANOTHER REPAIR MECHANISM :
 In another type of repair mechanism, nucleotide excision repair, enzymes replace
incorrect bases by making a cut on both the 3′ and 5′ ends of the incorrect base.
 The segment of DNA is removed and replaced with the correctly-paired
nucleotides by the action of DNA pol.
 Once the bases are filled in, the remaining gap is sealed with a phosphodiester
linkage catalyzed by DNA ligase.
 This repair mechanism is often employed when UV exposure causes the formation
of pyrimidine dimers.