Download MBBS Biochemistry PPT 20 Replication Lecture Notes

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1

Objectives

Steps of DNA Synthesis

DNA polymerases in prokaryotes and Eukaryotes

Difference in function of topoisomerase and ligase

Replication inhibitors

DNA repair

Action of Telomerase

2
An 8-year-old female, born out of the second

degree consanguineous marriage presented
with complaints of hypo- and hyper-
pigmented spots since the age of 1 year all over
the body which gradually increased in size and
number associated with small warty growths
over face for 5 years. She had a history of acute
sunburn all over the body

Vora et al 2016

3

except armpits and perineum after sun exposure.

The patient also had photophobia with redness,
watering of eyes. On examination, she had
multiple hyperpigmented warty growths over
face [Figure 1]. The patient was advised biopsy
and photoprotection, but parents denied biopsy.
After 1 year, patient came with an increase in
number, size of the growths. All the growths
were, warty and firm in consistency [Figure 1b].

4
A nontender nodular growth measuring about

2 cm ? 3 cm was present over the right
mandibular region from which biopsy was
taken which was suggestive of nodular
melanoma [Figure 2]. Another biopsy from
growth over right temporal region showed
features of hypertrophic type of actinic
keratosis . Routine investigations were within
normal limits.

5

The patient was referred to oncology

department, but relatives refused for any
further management. The patient visited our
department again after 2 years of the first visit
with small fungating growths over face, scalp
(4 cm ? 4 cm) and large foul smelling fungating
growth over nape of the neck extending over to
upper back (10 cm ? 8 cm) [Figure 3].

6
Biopsy was taken from the growth over the

nape of the neck which showed features of
squamous cell carcinoma [Figure 3]. There was
a history of injury to the right eye, in between
the two visits for which evisceration was done.
The left eye was congested; right orbital socket
was empty

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What is the likely diagnosis?

How would you make a definite diagnosis?

What is the pathophysiology of this disorder?

11

What is replication?

Biologic process of producing 2 identical
replicas of DNA from one original DNA
molecule.

Purpose :

Provision of progeny with genetic information

12
First enzymologic observations on DNA

replication were made by

Arthur Kornberg

Existence of replication enzyme

DNA polymerase I

13

Steps Involved in DNA Replication in Eukaryotes

1. Identification of the origins of replication
2. ATP hydrolysis-driven unwinding of dsDNA to
provide an ssDNA template
3. Formation of the replication fork; synthesis of
RNA primer
4. Initiation of DNA synthesis and elongation
5. Formation of replication bubbles with ligation of
the newly synthesized DNA segments
6. Reconstitution of chromatin structure

14
Origin of DNA replication

Adopted from Harper' text books

15

Unwinding of DNA

Adopted from Harper' text books

16
Formation of Replication fork: 4 components

Helicase
Primase
DNA pol
SSB

Adopted from Harper' text books

17

Classes of Proteins Involved in Replication

Protein

Function

DNA polymerases

Deoxynucleotide polymerization

Helicases

ATP -driven processive unwinding of
DNA

Topoisomerases

Relieve torsional strain that results
from
helicase-induced unwinding

DNA primase

Initiates synthesis of RNA primers

Single-strand binding Prevent premature reannealing of
proteins (SSBs)

dsDNA

DNA ligase

Seals the single strand nick between
the
nascent chain and Okazaki fragments
on
lagging strand

18
Chain elongation

Rate at which polymerisation

Processivity

Number of nucleotides added to the nacsent

chainbefore the polymerase disengages from
template

Proof reading

Identify copying errors

19

Initiation and Elongation
of DNA synthesis

The initiation of DNA synthesis
upon a primer of RNA
The RNA-primed synthesis of DNA
demonstrating the template function of
the
complementary strand of parental DNA

21

The generation of "replication
bubbles" during the process of
DNA synthesis

E coli genome: 5X10^6 bp

How to solve?

Replication rate: 3X10^5 bp/min
How long = 30 min

1. Bideirectional
2. Multiple origins

Mammalian genome: 3X10^9 bp

Adopted from Harper' text books

22
Supercoiling of DNA

23

24
Reactions catalyzed by

DNA topoisomerase I

25

Should be as few error as possible

3' to 5' exonuclease activity

26
Removal of RNA primer and filling of the resulting "gaps"

by DNA polymerase I

Adopted from Lippincott's' text books

27

Action of DNA ligase:
Formation of a
phosphodiester bond

28
Rate of polymerization in eukaryotic cells

compared to prokaryotes.....?

Nuclear organization and chromatin structure

determines the regulation and initiation of
DNA synthesis

Replicated DNA assembled into nucleosome

and histone octamers distributed to each arm
of the replication fork

Facilitated by histone chaperone proteins

working in concert with chromatin remodelling
complexes

29

Comparison of two types of
nick-sealing reactions on DNA

Adopted from Harper' text books
Functionally similar proteins

Prokaryote

Eukaryote

function

Hexameric Dna

Hexameric

Unwinds the DNA

complex

minichromosomal
maintenance (MCM)
complex

SSB

Replication protein A

Prevent reannealing

DnaG

Pol

Primase synthesizes RNA
primer

Pol III

Pol (leading) and Pol

Processive, leading and lagging

(Lagging)

strand synthesis

Pol I

Rnase H and FEN1 (flap of

RNA primers removed

endonuclease)

Pol II

DNA repair

Pol I

Gap filling following DNA
replication,
repair, and recombination

Mitochondrial DNA synthesis

subunit of Pol III

PCNA (Proliferating cell

Sliding clamp for high

nuclear antigen)

procesivity



A 21-year-old college student presents to the clinic complaining of

a sudden onset of chills and fever, muscle aches, headache,

fatigue, sore throat, and painful nonproductive cough 3 days prior

to fall final exams. Numerous friends of the patient in the

dormitory reported similar symptoms and were given the

diagnosis of influenza. He said that some of them were given a

prescription for ribavirin. On examination, he appears ill with

temperature 39.4?C (103?F). His skin is warm to the touch, but no

rashes are appreciated. The patient has mild cervical lymph node
enlargement but otherwise has a normal examination.

What is the most likely diagnosis?



What is the biochemical mechanism of action of ribavirin?

32
33

Inhibitors of DNA
Replication

34
Inhibitors of DNA Replication

Antibacterial agents

Action

Ciprofloxacin

Bacterial DNA gyrase

Nalidixic acid

Bacterial DNA gyrase

Anticancer agents

Adriamycin

Human topoisomerase

Etoposide

Human topoisomerase

6-mercaptopurine

Human DNA polymerase

5-fluoro uraci

Thymidylate synthase

Cytosine arabinosidede

Nucleoside analog

(Cytarabine)

Anti viral agent

adenine arabinoside

Nucleoside analog

(Vidarabine)

35

Each strand of the double helix serves as a template

(semiconservative replication).

occurs in the S phase of the cell cycle and begins at

the origin of replication.

strands are separated locally, forming replication

forks.

Replication of dsDNA is bidirectional

Helicase unwinds the double helix.

As the two strands of the double helix are separated,
supercoils are produced
DNA topoisomerases Types I and II remove supercoils

36


The primer for de novo DNA synthesis is a short stretch of RNA

synthesized by primase



DNA polymerases synthesize new DNA strands only in the 5'3`

direction.



one of the newly synthesized stretches of

nucleotide chains must grow in the 5'3' direction toward the replication

fork (leading strand)



In E. coli DNA chain elongation is catalyzed by DNA polymerase III



The enzyme "proofreads" the newly synthesized DNA with its 3'5'

exonuclease activity.



RNA primers are removed by DNA polymerase I, using its 5'3'

exonuclease activity.



The final phosphodi-ester linkage is catalyzed by DNA ligases



at least five eukaryotic DNA polymerase



Nucleoside analogs containing modified sugars can be used to

block DNA chain growth.

37

Video on DNA Replication

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Multiple DNA repair pathways of variable accuracy

0
1. Methyl-directed mismatch repair

41

Aadopted from Lippincott's text book

Methyl-directed mismatch repair

contd

42

Aadopted from Lippincott's text book
2. Nucleotide excision repair

43

Aadopted from Lippincott's text book

Patient with xeroderma pigmentosum

44

Aadopted from Lippincott's text book
inherited as an autosomal recessive trait

characterized

by photosensitivity, pigmentary changes,
premature skin ageing and malignant tumour
development

defect in DNA repair (NER)

45

3. Base excision repair

46

Aadopted from Lippincott's text book
Correction of base
alterations by base
excision repair

47

Aadopted from Lippincott's text book

4. The multistep
mechanism of

DNA double-strand

break repair

Aadopted from Harper's text book
Clinical implications

49

Human Diseases of DNA Damage Repair

Defective Nonhomologous End Joining Repair (NHEJ)

Severe combined immunodeficiency disease (SCID)
Radiation sensitive severe combined
immunodeficiency disease (RS-SCID)

Defective Homologous Repair (HR)

Bloom syndrome (BS)
Werner syndrome (WS)
Breast cancer suspectibility 1 and 2 (BRCA1, BRCA2
AT -like disorder (AT LD)

50
Human Diseases of DNA Damage Repair

Defective DNA Nucleotide Exicision Repair (NER)
Xeroderma pigmentosum (XP)
Cockayne syndrome (CS)
Trichothiodystrophy (TT D)

Defective DNA Base Excision Repair (BER)
MUTYH-associated polyposis (MAP)

Defective DNA Mismatch Repair (MMR)
Hereditary nonpolyposis colorectal cancer (HNPCC)

51



If a double-stranded DNA molecule undergoes two rounds of

replication in an in vitro system that contains all of the necessary

enzymes and

nucleoside triphosphates that have been labeled with 32P, which

of the

following best describes the distribution of radioactivity in the

four

resulting DNA molecules?

A. Exactly one of the molecules contains no radioactivity.

B. Exactly one of the molecules contains radioactivity in only one

strand.

C. Two of the molecules contain radioactivity in both strands.

D. Three of the molecules contain radioactivity in both strands.

E. All four molecules contain radioactivity in only one strand.

52
A 48-year-old man has had a lengthy history of

skin cancer. In the past 6 years he has had over 30

neoplasms removed from sun-exposed areas and

has been diagnosed with xeroderma

pigmentosum. Which of the following best

describes the enzymatic defect in patients with

xeroderma pigmentosum?

A. DNA polymerase

B. DNA polymerase

C. DNA ligase

D. Excision repair enzymes

E. RNA polymerase III

53

A 10-year-old girl is brought to the dermatologist by

her parents. She has many freckles on her face,

neck, arms, and hands, and the parents report that

she is unusually sensitive to sunlight. Two basal cell

carcinomas are identified on her face. Which of the

following processes is most likely to be defective in

this patient?

A. Repair of double-strand breaks.

B. Removal of mismatched bases from the 3'-end of

Okazaki fragments.

C. Removal of pyrimidine dimers from DNA.

D. Removal of uracil from DNA.

54
55

Several thousand tandem repeats of GT rich

hexamer (AGGGTT) that cap the ends of
eukaryotic chromosomes that help stabilize the
chromosome., preventing attack by nucleases

Telomeres (Greek telos, "end") : complexes of

DNA plus proteins (Shelterin)

To accommodate the wastage that occurs

during replication

Allow repair system to distinguish

56
Organisms are able to generate progeny that

contain full length telomeres .

How is it possible?

Telomerase

Ribonucleoprotein expressed in germ cells, stem cells

, most cancer cells

NOT in SOMATIC cells

Replicative senescence

57

Mechanism of action of telomerase

58

Aadopted from Lippincott's text book
Mechanism of action of telomerase

contd

59

Aadopted from Lippincott's text book

Mechanism of action of telomerase

contd

60

Aadopted from Lippincott's text book
Telomerase

Transposons

Most transposons transcribed into RNA which is

used as a template for DNA synthesis by a reverses
transcriptase encoded by transposons and the DNA
is randomly inserted into the genome

Called retrotransposon or retroposon

61

Telomeres are complexes of DNA and protein that protect

the ends of linear chromosomes. In most normal human

somatic cells, telomeres shorten with each division. In stem

cells and in cancer cells, however, telomeric length is

maintained. In the synthesis

of telomeres:

A. telomerase, a ribonucleoprotein, provides both the

RNA and the polymerase needed for synthesis.

B. the RNA of telomerase serves as a primer.

C. the polymerase of telomerase is a DNA-directed

DNA polymerase.

D. the shorter, 3'5' strand gets extended.

E. the direction of synthesis is 3'5'.

62
The eukaryotic cell cycle

63

Aadopted from Lippincott's text book

During the S phase, the nuclear DNA is

completely replicated once and only once.

Once chromatin has been replicated, it is marked

so as to prevent its further replication
until it again passes through mitosis.

molecular mechanisms

dissociation and/or cyclin-CDK phosphorylation
and subsequent degradation of several origin binding

proteins

origins fire only once per cell cycle.

64
Progress through the mammalian cell cycle is
continuously monitored via multiple cell-cycle
checkpoints

Cyclins and Cyclin-Dependent Kinases

Involved in Cell-Cycle Progression

Cyclin

Kinase

Function

D

CDK4, CDK6

Progression past
restriction
point at G1/S
boundary

E, A

CDK2

Initiation of DNA
synthesis in
early S phase

B

CDK1

Transition from
G2 to M

66
Points during which the indicated cyclins and cyclin-dependent
kinases are activated

This post was last modified on 05 April 2022