DNA polymerases in prokaryotes and Eukaryotes
Difference in function of topoisomerase and ligase
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Replication inhibitors
DNA repair
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Action of Telomerase2
An 8-year-old female, born out of the second
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degree consanguineous marriage presentedwith 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
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over face for 5 years. She had a history of acutesunburn all over the body
Vora et al 2016
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3except armpits and perineum after sun exposure.
The patient also had photophobia with redness,
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watering of eyes. On examination, she hadmultiple 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
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number, size of the growths. All the growthswere, warty and firm in consistency [Figure 1b].
4
A nontender nodular growth measuring about
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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
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growth over right temporal region showedfeatures of hypertrophic type of actinic
keratosis . Routine investigations were within
normal limits.
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5The patient was referred to oncology
department, but relatives refused for any
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further management. The patient visited ourdepartment 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
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upper back (10 cm ? 8 cm) [Figure 3].6
Biopsy was taken from the growth over the
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nape of the neck which showed features ofsquamous 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
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was empty7
8
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910
What is the likely diagnosis?
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How would you make a definite diagnosis?What is the pathophysiology of this disorder?
11
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What is replication?
Biologic process of producing 2 identical
replicas of DNA from one original DNA
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molecule.Purpose :
Provision of progeny with genetic information
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12
First enzymologic observations on DNA
replication were made by
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Arthur Kornberg
Existence of replication enzyme
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DNA polymerase I13
Steps Involved in DNA Replication in Eukaryotes
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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
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RNA primer4. Initiation of DNA synthesis and elongation
5. Formation of replication bubbles with ligation of
the newly synthesized DNA segments
6. Reconstitution of chromatin structure
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14
Origin of DNA replication
Adopted from Harper' text books
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15
Unwinding of DNA
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Adopted from Harper' text books16
Formation of Replication fork: 4 components
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HelicasePrimase
DNA pol
SSB
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Adopted from Harper' text books17
Classes of Proteins Involved in Replication
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Protein
Function
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DNA polymerasesDeoxynucleotide polymerization
Helicases
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ATP -driven processive unwinding of
DNA
Topoisomerases
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Relieve torsional strain that results
from
helicase-induced unwinding
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DNA primaseInitiates synthesis of RNA primers
Single-strand binding Prevent premature reannealing of
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proteins (SSBs)dsDNA
DNA ligase
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Seals the single strand nick between
the
nascent chain and Okazaki fragments
on
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lagging strand18
Chain elongation
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Rate at which polymerisationProcessivity
Number of nucleotides added to the nacsent
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chainbefore the polymerase disengages from
template
Proof reading
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Identify copying errors
19
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Initiation and Elongationof DNA synthesis
The initiation of DNA synthesis
upon a primer of RNA
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The RNA-primed synthesis of DNAdemonstrating the template function of
the
complementary strand of parental DNA
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21The generation of "replication
bubbles" during the process of
DNA synthesis
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E coli genome: 5X10^6 bp
How to solve?
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Replication rate: 3X10^5 bp/minHow long = 30 min
1. Bideirectional
2. Multiple origins
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Mammalian genome: 3X10^9 bp
Adopted from Harper' text books
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22Supercoiling of DNA
23
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24Reactions catalyzed by
DNA topoisomerase I
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25Should be as few error as possible
3' to 5' exonuclease activity
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26
Removal of RNA primer and filling of the resulting "gaps"
by DNA polymerase I
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Adopted from Lippincott's' text books
27
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Action of DNA ligase:Formation of a
phosphodiester bond
28
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Rate of polymerization in eukaryotic cellscompared to prokaryotes.....?
Nuclear organization and chromatin structure
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determines the regulation and initiation of
DNA synthesis
Replicated DNA assembled into nucleosome
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and histone octamers distributed to each arm
of the replication fork
Facilitated by histone chaperone proteins
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working in concert with chromatin remodelling
complexes
29
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Comparison of two types of
nick-sealing reactions on DNA
Adopted from Harper' text books
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Functionally similar proteinsProkaryote
Eukaryote
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function
Hexameric Dna
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HexamericUnwinds the DNA
complex
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minichromosomal
maintenance (MCM)
complex
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SSBReplication protein A
Prevent reannealing
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DnaG
Pol
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Primase synthesizes RNAprimer
Pol III
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Pol (leading) and PolProcessive, leading and lagging
(Lagging)
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strand synthesis
Pol I
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Rnase H and FEN1 (flap ofRNA primers removed
endonuclease)
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Pol II
DNA repair
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Pol IGap filling following DNA
replication,
repair, and recombination
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Mitochondrial DNA synthesis
subunit of Pol III
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PCNA (Proliferating cellSliding clamp for high
nuclear antigen)
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procesivity
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A 21-year-old college student presents to the clinic complaining ofa sudden onset of chills and fever, muscle aches, headache,
fatigue, sore throat, and painful nonproductive cough 3 days prior
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to fall final exams. Numerous friends of the patient in the
dormitory reported similar symptoms and were given the
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diagnosis of influenza. He said that some of them were given aprescription for ribavirin. On examination, he appears ill with
temperature 39.4?C (103?F). His skin is warm to the touch, but no
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rashes are appreciated. The patient has mild cervical lymph node
enlargement but otherwise has a normal examination.
What is the most likely diagnosis?
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What is the biochemical mechanism of action of ribavirin?
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3233
Inhibitors of DNA
Replication
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34
Inhibitors of DNA Replication
Antibacterial agents
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Action
Ciprofloxacin
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Bacterial DNA gyraseNalidixic acid
Bacterial DNA gyrase
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Anticancer agents
Adriamycin
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Human topoisomeraseEtoposide
Human topoisomerase
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6-mercaptopurine
Human DNA polymerase
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5-fluoro uraciThymidylate synthase
Cytosine arabinosidede
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Nucleoside analog
(Cytarabine)
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Anti viral agentadenine arabinoside
Nucleoside analog
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(Vidarabine)
35
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Each strand of the double helix serves as a template(semiconservative replication).
occurs in the S phase of the cell cycle and begins at
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the origin of replication.
strands are separated locally, forming replication
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forks.Replication of dsDNA is bidirectional
Helicase unwinds the double helix.
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As the two strands of the double helix are separated,
supercoils are produced
DNA topoisomerases Types I and II remove supercoils
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36The primer for de novo DNA synthesis is a short stretch of RNA
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synthesized by primaseDNA polymerases synthesize new DNA strands only in the 5'3`
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direction.
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one of the newly synthesized stretches ofnucleotide chains must grow in the 5'3' direction toward the replication
fork (leading strand)
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In E. coli DNA chain elongation is catalyzed by DNA polymerase III
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The enzyme "proofreads" the newly synthesized DNA with its 3'5'
exonuclease activity.
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RNA primers are removed by DNA polymerase I, using its 5'3'
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exonuclease activity.The final phosphodi-ester linkage is catalyzed by DNA ligases
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at least five eukaryotic DNA polymerase
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Nucleoside analogs containing modified sugars can be used to
block DNA chain growth.
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37
Video on DNA Replication
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3839
Multiple DNA repair pathways of variable accuracy
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01. Methyl-directed mismatch repair
41
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Aadopted from Lippincott's text bookMethyl-directed mismatch repair
contd
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42
Aadopted from Lippincott's text book
2. Nucleotide excision repair
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43
Aadopted from Lippincott's text book
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Patient with xeroderma pigmentosum44
Aadopted from Lippincott's text book
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inherited as an autosomal recessive traitcharacterized
by photosensitivity, pigmentary changes,
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premature skin ageing and malignant tumourdevelopment
defect in DNA repair (NER)
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453. Base excision repair
46
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Aadopted from Lippincott's text book
Correction of base
alterations by base
excision repair
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47
Aadopted from Lippincott's text book
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4. The multistepmechanism of
DNA double-strand
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break repairAadopted from Harper's text book
Clinical implications
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49Human Diseases of DNA Damage Repair
Defective Nonhomologous End Joining Repair (NHEJ)
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Severe combined immunodeficiency disease (SCID)
Radiation sensitive severe combined
immunodeficiency disease (RS-SCID)
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Defective Homologous Repair (HR)Bloom syndrome (BS)
Werner syndrome (WS)
Breast cancer suspectibility 1 and 2 (BRCA1, BRCA2
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AT -like disorder (AT LD)50
Human Diseases of DNA Damage Repair
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Defective DNA Nucleotide Exicision Repair (NER)Xeroderma pigmentosum (XP)
Cockayne syndrome (CS)
Trichothiodystrophy (TT D)
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Defective DNA Base Excision Repair (BER)MUTYH-associated polyposis (MAP)
Defective DNA Mismatch Repair (MMR)
Hereditary nonpolyposis colorectal cancer (HNPCC)
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51
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If a double-stranded DNA molecule undergoes two rounds ofreplication in an in vitro system that contains all of the necessary
enzymes and
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nucleoside triphosphates that have been labeled with 32P, which
of the
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following best describes the distribution of radioactivity in thefour
resulting DNA molecules?
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A. Exactly one of the molecules contains no radioactivity.
B. Exactly one of the molecules contains radioactivity in only one
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strand.C. Two of the molecules contain radioactivity in both strands.
D. Three of the molecules contain radioactivity in both strands.
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E. All four molecules contain radioactivity in only one strand.
52
A 48-year-old man has had a lengthy history of
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skin cancer. In the past 6 years he has had over 30
neoplasms removed from sun-exposed areas and
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has been diagnosed with xerodermapigmentosum. Which of the following best
describes the enzymatic defect in patients with
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xeroderma pigmentosum?
A. DNA polymerase
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B. DNA polymeraseC. DNA ligase
D. Excision repair enzymes
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E. RNA polymerase III
53
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A 10-year-old girl is brought to the dermatologist byher parents. She has many freckles on her face,
neck, arms, and hands, and the parents report that
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she is unusually sensitive to sunlight. Two basal cell
carcinomas are identified on her face. Which of the
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following processes is most likely to be defective inthis patient?
A. Repair of double-strand breaks.
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B. Removal of mismatched bases from the 3'-end of
Okazaki fragments.
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C. Removal of pyrimidine dimers from DNA.D. Removal of uracil from DNA.
54
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55Several thousand tandem repeats of GT rich
hexamer (AGGGTT) that cap the ends of
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eukaryotic chromosomes that help stabilize thechromosome., preventing attack by nucleases
Telomeres (Greek telos, "end") : complexes of
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DNA plus proteins (Shelterin)To accommodate the wastage that occurs
during replication
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Allow repair system to distinguish
56
Organisms are able to generate progeny that
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contain full length telomeres .
How is it possible?
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TelomeraseRibonucleoprotein expressed in germ cells, stem cells
, most cancer cells
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NOT in SOMATIC cells
Replicative senescence
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57Mechanism of action of telomerase
58
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Aadopted from Lippincott's text book
Mechanism of action of telomerase
contd
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59
Aadopted from Lippincott's text book
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Mechanism of action of telomerasecontd
60
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Aadopted from Lippincott's text book
Telomerase
Transposons
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Most transposons transcribed into RNA which is
used as a template for DNA synthesis by a reverses
transcriptase encoded by transposons and the DNA
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is randomly inserted into the genomeCalled retrotransposon or retroposon
61
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Telomeres are complexes of DNA and protein that protect
the ends of linear chromosomes. In most normal human
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somatic cells, telomeres shorten with each division. In stemcells and in cancer cells, however, telomeric length is
maintained. In the synthesis
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of telomeres:
A. telomerase, a ribonucleoprotein, provides both the
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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
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DNA polymerase.
D. the shorter, 3'5' strand gets extended.
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E. the direction of synthesis is 3'5'.62
The eukaryotic cell cycle
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63Aadopted from Lippincott's text book
During the S phase, the nuclear DNA is
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completely replicated once and only once.
Once chromatin has been replicated, it is marked
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so as to prevent its further replicationuntil it again passes through mitosis.
molecular mechanisms
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dissociation and/or cyclin-CDK phosphorylationand subsequent degradation of several origin binding
proteins
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origins fire only once per cell cycle.64
Progress through the mammalian cell cycle is
continuously monitored via multiple cell-cycle
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checkpointsCyclins and Cyclin-Dependent Kinases
Involved in Cell-Cycle Progression
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Cyclin
Kinase
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FunctionD
CDK4, CDK6
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Progression past
restriction
point at G1/S
boundary
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E, A
CDK2
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Initiation of DNAsynthesis in
early S phase
B
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CDK1
Transition from
G2 to M
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66
Points during which the indicated cyclins and cyclin-dependent
kinases are activated
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