MBBS Biochemistry

1. DNA REPLICATION

2. DNA :The Genetic Material
• DNA is macromolecule that carries
genetic information from
generation to generation.
• DNA – Reserve Bank of Genetic
Information.

3. • Made up of subunits called
nucleotides
• Nucleotide made of:
1. Phosphate group
2. 5-carbon sugar
3. Nitrogenous base

4. DNA STRUCTURE

7. Replication - DNA copies itself
exactly (Occurs within the
nucleus)
 Any mistake in copying -
Mutation
 DNA mutation - chromosomal
mutation

8. Basic rules of
Replication
A. Semi-conservative
B. Starts at the ‘origin’
C. Synthesis always in the 5-3’
direction
D. Can be uni or bidirectional
E. Semi-discontinuous
F. RNA primers required
G. During S-phase of cell cycle

9. DNA
Replication

10. One strand of duplex
passed on unchanged
to each of the
daughter cells. This
'conserved' strand acts
as a template for the
synthesis of a new,
complementary strand
by the enzyme DNA
polymerase

11. Basic requirement of DNA Replication
• Substrate
• Template
• Primer
• Enzyme
• Protein

12. Substrate – The Four
deoxyribonucleoside triphosphate:
1. dATP
2. dCTP
3. dGTP
4. dTTP
Template - Each strand within the
parent double helix, acts as a template.
Primer – Short piece of RNA having a
free 3’end
(about 5-50 nucleotides)

13. Enzymes & Protein
Prokaryotes
• DNA A Protein
• Helicase (DNA B)
• SSBs
• DNA Topoisomerase
• Primase
• DNA Polymerase |,
||, & |||
• DNA Ligase
Eukaryotes
• DNA A Protein
• Helicase (DNA B)
• SSBs
• DNA Topoisomerase
• Primase
• DNA Polymerase
alpha & delta
• DNA Ligase

14. •Helicase:-
• unwinds the parental DNA
• Form replication fork
• SSBs Protein:-
• Binds with single stranded DNA
• Stabilizes the separated and
prevent renaturation of DNA

15. • DNA TOPOISOMERASE:-
Type 1 - cut the single DNA strand to
overcome the
problem of super coils and reseals the
strand
Type 2- also known as DNA GYRASE
-cut both strand and
reseal.
• PRIMASE:- a specific RNA Polymerase
produce a RNA PRIMER.

16. • IN PROKERYOTES :-
1.DNA POLYMERASE - I
2.DNA POLYMERASE -
II
3.DNA POLYMERASE -
III
DNA Polymerase

17. DNA POLYMERASE -I
- Polymerization activity-5’-3’
- Exonuclease activity [proof reading
activity] -3’-5’ (also in 5’-3’)
-RNA dependent DNA polymerization
activity
- Replace RNA primer and add DNA
DNA POLYMERASE -II
- Polymerization activity -5’-3’
- Exonuclease activity -3’-5’
Backup enzyme- mostly in repair

18. DNA POLYMERASE-
III
-Polymerization activity – 5’-3’
- Exonuclease activity – 3’-5’
- Lagging and leading strand synthesis
- Clamp loader region
- Hold DNA strand

19. DIFFERENT TYPES OF EUKARYOTIC
POLYMERASES
NAME FUNCTION
DNA polymerase alpha Initiator polymerase. It has
primase activity to synthesize
RNA primer. No exonuclease
activity
DNA polymerase beta DNA repair
DNA polymerase gama Mitochondria DNA synthesis. No
exonuclease activity
DNA polymerase delta Primary enzyme of DNA
synthesis
DNA polymerase epsilon DNA proof reading and DNA
repair

20. Eukaryotic DNA Polymerase

22. RNA primer
Synthesized by RNA Primase
One for leading strand
Multiple for lagging strand

23. Mechanism of DNA
replication:-

24.  Initiation:-
• Proteins bind to DNA and open up
double helix.
• Prepare DNA for complementary base
pairing.
Elongation:-
• Proteins connect the correct
sequences of nucleotides into a
continuous new strand of DNA.
Termination:-
• Proteins release the replication
complex.

25. Initiation
 Begins at origin of
replication(ori)
• Prokaryotes – single origin site
• Eukaryotes – multiple origin site
Unwinding of DNA forms
replicating fork (V shaped
region) where active synthesis
occur

26. Uni or bidirectional
Replication forks move in one or opposite directions

28. Helicase unwinds the 2 strand
of the DNA utilizes energy of
ATP hydrolysis.
The Stress produced by
supercoiling (or unwinding) is
released by topoisomerases
Cut one or both strands of
DNA.
SSB stabilizes the separated
strand and prevent

30. Topoisomerase

31. SSB binds to single strand
Released by Topoisomerase
Stress produce due to super coiling
Formation of &Replicating Bubble
Unwinding of DNA Strand by Helicase
Starts at origin of replication

32. Synthesis is ALWAYS in the 5’-3’ direction
• One strand is synthesize continuously
while other in fragments
Semi-discontinuous replication

33. • Leading Strand– The DNA strand which
runs in 3’ to 5’ direction is
synthesized in continuous manner
• point of origin toward the opening
replication fork

34. • Lagging Strand– The DNA strand
which runs in 5’ to 3’ direction is
synthesized in discontinuous manner.
• This strand is made in MANY short
segments k/a OKAZAKI FRAGMENTS.

37. TERMINATION
• The enzyme Ligase joins the
Okazaki fragments together to
make one strand.

39. DNA Ligase

42. • Origin of replication is identified. Then unwinding of parental
DNA to form a replication fork.
• RNA primer complementary to the DNA template is synthesized
by RNA primase.
• DNA synthesis is continuous in the leading strand (toward replication
fork) by DNA polymerase.
• DNA synthesis is discontinuous in the lagging strand (away from the
fork), as Okazaki fragments.
• Elongation: In both strands, the synthesis is from 5' to 3‘ direction.
• Then the RNA pieces are removed; the gaps are filled by
deoxynucleotides by DNAP and the pieces are ligated by DNA ligase.
• Proofreading is done by the DNA polymerase.
• Finally organized into chromatin.
• Main enzymes involved in replication are: DNA polymerases, helicases,
topoisomerases, DNA primase, single-strand binding proteins, and
DNA ligase.
Summary of DNA Replication

44. Prokaryotes Eurokaryotes
DNA Circular DNA Linear
Origin Origin of replication Replication at multiple sites
Single strand
binding
proteins
Co-operative
binding to the SS
DNA
SS DNA binding proteins bind
at the replication fork.
RNA primer Required for
synthesis of both
strands
DNAP alpha has primase
activity and initiates synthesis
of both lagging and leading
strands
DNA
polymerases
Major polymerizing
enzyme is DNAP–III
Major polymerizing enzymes
are DNAP delta and DNAP
epsilon.
Comparison of features of Replication in Prokaryotic and
Eukaryotic Cells

45. Prokaryotes Eurokaryotes
Proof-
reading
DNAP III also has 3’
to 5’ exonuclease
activity so that any
wrong base is
removed and the
correct one added
DNAP delta and epsilon both
have 3’→ 5’ exonuclease
activity and therefore serve
the proofreading function
Gap filling The RNA primer is
removed and gap
filled by DNAP
RNA primer is removed by
RNAse H and FEN1.
Polymerase beta is involved in
gap filling and DNA repair.
Inhibitors Ciprofloxacin and
Novobiocin inhibit
topoisomerase
Etoposide, Adriamycin and
Camptothecin inhibit
Topoisomerase
Comparison of features of Replication in Prokaryotic and
Eukaryotic Cells

46. DNA INHIBITORS
o Many antibacterial & antiviral inhibit
replication drugs & many chemotherapeutic
drugs inhibit replication.
o Many antibacterial drugs inhibit prokaryotic
enzymes, topoisomerase II (DNA gyrase)
much more than the eukaryotic one & are
widely used for the treatment of urinary tract
& other infections including those due to
bacillus antharacis(ANTHRAX).

47. Camptothecin an antitumor drug
inhibits human topoisomerase-I.
Certain anticancer and antivirus drugs
inhibits elongation by incorporating
certain nucleotide analogs.
NOVOBIOCIN the binding of ATP
to gyrase which requires for its
activity.
NALIDIXIC & CIPROFLAXIN , in
contrast, interferes with the
breakage & rejoining of DNA chains.

49. Drug Action (inhibition of)
Antibacterial agents
Ciprofloxacin Bacterial DNA gyrase
Nalidixic acid do
Novobiocin do
Anticancer agents
Etoposide Human topoisomerase
Adriamycin do
Doxorubicin do
6-mercaptopurine Human DNA polymerase
5-fluorouracil Thymidylate synthase
Inhibitors of DNA Replication

50. DNA Organization

51. DNA damage & REPAIR
• Despite proof
reading and
mismatch repair
during
replication some
mismatched
bases do
persist.

52. Mechanism for DNA Repair involve
• First recognition of distorted region of
the DNA.
• Removal of the damaged region of the
DNA strand.
• Then filling of gap left by the removal
of the damaged DNA by DNA
polymerase.
 Sealing the nick in the strand that has
undergone repair by a ligase.

53. • Oxidation of bases (e.g. 8-oxo-7,8-dihydroguanine).
• Alkylation (methylation) of bases, such as 7-methyl guanosine,
1-methyl adenine, 6-methyl guanine.
• Hydrolysis of bases, such as deamination, depurination, and
depyrimidination.
• Adduct formation, e.g. benzo[a]pyrene diol epoxide causes dG adduct.
• Mismatch of bases, due to errors in DNA replication.
• Monoadduct damage cause by change in single nitrogenous base of DNA.
• Ultraviolet-B (UV-B) light causes cross-linking between adjacent cytosine
and thymine bases creating pyrimidine dimers. This is a direct damage.
• Ultraviolet-A light creates mostly free radicals, causing indirect DNA
damages.
• Ionizing radiation such as gamma-rays may induce irreparable DNA damage.
• Elevated temperature causes depurination (loss of purine bases from the
DNA backbone) and single strand breaks.
• Chemicals such as aromatic hydrocarbons cause DNA adducts.
Types of Damage

54. Causes of DNA damage
Causes Agents
Replication
errors
1 base pair error in 1010 base pair per cycle
Chemicals Insecticides, pesticides, gases, Food adulterants , preservatives
Deamination Spontaneous, Nitrates, Bisulphates
Alkylation Dimethylsulphate and nitrogen mustard
Depurination Acidic condition
Thymine dimers UV rays
Chain break Xrays, Gamma rays
Free Radicals H2O2, Hydroxyl radicals
Adduct (
Alteration)
Formation
Cigarette smoking, benzopyrines

55. • Mismatch repair
• Base excision repair
• Nucleotide excision repair
• Double strand break repair
Types of DNA Repair

56. Mechanism Defect Repair
Mismatch
repair
Copying error 1-5
bases unpaired
Strand cutting,
exonuclease
digestion
Nucleotide
exicision repair
(NER)
Chemical damage to
a segment
30 bases removed;
then correct bases
added
Base excision
repair
Chemical damage to
single base
Base removed by N-
glycosylase; new
base added
Double strand
break
Free radicals and
radiation
Unwinding,
alignment, ligation
DNA Repair Mechanisms

57. Exonucleolytic proofreading of
DNA polymerase.

58. Base Excision repair
• Deamination
• U removed by glycosylase
• Cut by endonuclease
• Lyase removes sugar and
phosphate
• Repair by polymerase and
ligase

59. Mismatch Repair

60. HNPCC
• Mutation to the proteins involved in mismatch repair in
humans is associated with hereditary nonpolyposis colorectal
cancer (HNPCC), also known as Lynch syndrome.
• With HNPCC, there is an increased risk for developing colon
cancer (as well as other cancers), but only about 5% of all
colon cancer is the result of mutations in mis- match repair.

61. Nucleotide excision repair
Pyrimidine Dimers due to
UVrays
Corrected by exinuclease

62. UV radiation and cancer
• Pyrimidine dimers can be formed in the skin cells of humans
exposed to unfiltered sunlight.
• In the rare genetic disease xeroderma pigmentosum (XP), the
cells cannot repair the damaged DNA, resulting in extensive
accumulation of mutations and, consequently, early and
numerous skin cancers
• XP can be caused by defects in any of the several genes that
code for the XP proteins required for nucleotide excision
repair of UV damage in humans.

63. Source: Lippincott’s textbook of Biochemistry

64. Double stand Break Repair
• High-energy radiation or oxidative free radicals can cause
double-strand breaks in DNA, which are potentially lethal to the
cell. Such breaks also occur naturally during gene
rearrangements.
• they are repaired by one of two systems.
• The first is nonhomologous end-joining repair, in which the ends
of two DNA fragments are brought together by a group of
proteins that effect their religation.
• However, some DNA is lost in the process. Consequently, this
mechanism of repair is error prone and mutagenic.
• Defects in this repair system are associated with a predisposition
to cancer and immunodeficiency syndromes.

65. Double stand Break Repair
• The second repair system, homologous
recombination repair, uses the enzymes that
normally perform genetic recombination
between homologous chromosomes during
meiosis. This system is much less error prone
than nonhomologous end-joining joining
because any DNA that was lost is replaced
using homologous DNA as a template.

66. • Xeroderma pigmentosum (XP): Defective nucleotide excision
repair (NER) mechanism; sensitivity to UV light; skin cancers.
• Ataxia telangiectasia (AT): Defective ATM gene; sensitivity to UV
light; lymphoreticular neoplasms.
• Fanconi anemia: Defective genes are in chromosomes 20q and
9q. Defect in DNA cross-link repair; increased occurrence of cancer.
• Bloom’s syndrome: Gene is in 15q. Defect is in DNA ligase or
helicase; lymphoreticular malignancies.
• Cockayne syndrome: Defect in NER mechanism; transcription
factor II H is defective; stunted growth and mental retardation.
• Hereditary polyposis colon cancer (Lynch syndrome): Defective
gene in chromosome 2. Defect in hMSH 1 and 2 genes;
mismatch repair is defective.
Diseases Associated with DNA Repair Mechanisms

67. • In eukaryotic replication , following
removal of RNA Primer from the
5’end of lagging strand ; a gap is
left.
• This gap exposes DNA strand to
attack of 5’ exonucleases.
• This problem is overcome by
Telomerase.

68. • Repeating, non-coding sequences at
the end of chromosome = protective
cap.
Telomeres

69. Action of Telomerase

70. Application of Telomere
Telomerase :-
Different level of
activity in
different cell.
• High in stem cell
and cancer
• Activity lost in old
age.
• Potential target
for newer
anticancer drugs.

71. Telomere and Telomerase
The replication always takes place from 5'
to 3' direction in the new strand. The DNA
polymerase enzyme is not able to synthesise the
new strand at the 5' end of the new strand. Thus, a
small portion (about 300 nucleotides) in the 3' ends of
the parent strands could not be replicated. This end
piece of the chromosome is called telomere.
Therefore, another enzyme, telomere terminal
transferase or telomerase takes up this job of
replication of the end piece of chromosomes.
Unless there is some mechanism to replicate
telomeres, the length of the chromosomes will go on
reducing at each cell division (gene loss). The
stability of the chromosome is thus lost. The
shortening of telomere end is prevented by an enzyme
telomerase. It contains an essential RNA component,
which provides the template for telomerase repeat
synthesis. Telomerase acts like a reverse
transcriptase. Telomerase recognises 3' end of
telomere, and then a small DNA strand is synthesised.

73. Reverse Transcriptase
RNA-directed DNA polymerases.
A reverse transcriptase is involved in the replication of
retroviruses, such as human immunodeficiency virus
(HIV)
These viruses carry their genome in the form of ssRNA
molecules.
Following infection of a host cell, the viral enzyme,
reverse transcriptase, uses the viral RNA as a template
for the 5'→3' synthesis of viral DNA, which then
becomes integrated into host chromosomes.
Reverse transcriptase activity is also seen with
transposons, DNA elements that can move about the
genome

74. Cell Cycle
The four phases of the cell cycle are G1, G2, S, and M. In G1 (gap 1) phase, the
cell prepares for DNA synthesis. DNA synthesis occurs during the S (synthesis)
phase of the cell cycle (Fig. 34.30). During the S phase, DNA is completely
replicated, but only once. Cell prepares for mitosis in G2 (gap 2) phase, when
proteins necessary for daughter cells are synthesized. Then the cell enters into the
M (mitotic) phase, when the chromosomes are visible under the microscope. The
whole cycle lasts about 24 hours; out of which M phase is only 1–2 hours. Those
cells which are not in division are said to be in G0 phase or resting phase.

75. References
• Textbook of Biochemistry, DM Vasudevan
• Textbook of Biochemistry, Lippincott