Download MBBS (Bachelor of Medicine, Bachelor of Surgery) 1st year (First Year) Biochemistry ppt lectures Topic 20 Replication Notes. - biochemistry notes pdf, biochemistry mbbs 1st year notes pdf, biochemistry mbbs notes pdf, biochemistry lecture notes, paramedical biochemistry notes, medical biochemistry pdf, biochemistry lecture notes 2022 ppt, biochemistry pdf.
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
7
8
9
10
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
38
39
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