DNA

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DNA

• DNA replication
• Prokaryotic cells
• Eukaryotic cells
• DNA repair

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Nucleoside
structure of deoxyadenosine
Nucleotide
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Structures of the bases
Purines
Pyrimidines
Thymine (T)
Adenine (A)
Guanine (G)
Cytosine (C)
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Ribose and Deoxyribose

• The sugar moiety of RNA differs from that of DNA.
• The difference is the presence of a 2 hydroxy
  gr. on the ribose molecule.

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Base Nucleoside (RNA) Deoxynucleoside
(DNA) Adenine Adenosine Deoxyadenosine Guanine
Guanosine Deoxyguanosine Cytosine Cytidine Deoxy
cytidine Uracil Uridine (not usually
found) Thymine (not usually found) (Deoxy)thymidi
ne
base
sugar
nucleoside
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RNA bases resemble those in DNA
Uracil in RNA, but Thymine in DNA
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ii). Structure of the DNA double helix
Structure of the DNA polynucleotide chain
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3

• polynucleotide chain
• 3,5-phosphodiester bond

phosphodiester linkage between 3'-OH group and
5'-phosphate groups
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A-T base pair
Hydrogen bonding of the bases
G-C base pair
Chargaffs rule The content of A equals the
content of T, and the content of G equals
the content of C in double-stranded DNA
from any species
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Central Dogma
DNA
Replication
Transcription
RNA
Translation
Protein
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Messelson and Stahl experiment
DNA Replication

• Semiconservative

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DNA replication is semi-conservative
Parental DNA strands
Each of the parental strands serves as a
template for a daughter strand
Daughter DNA strands
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Messelson and Stahl experiment

• Semi-conservative replication
• Daughter DNA contains one parental strand and one
  newly synthesized strand

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DNA Replication (prokaryotic cells)

• Initiation Initiation factors bind to the Ori ?
  unwinding a region of the DNA double helix
• Helicase unwinds the DNA double helix (use ATP)
• Single strands DNA binding proteins prevent ssDNA
  from double helical structure (dsDNA), keeping
  the helix open
• Topoisomerase I or DNA gyrase relieves twisting
  strain by cutting one strand. Topoisomerase II
  cut two DNA strand.

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Topoisomerase action during DNA replication
Fig. 6.9
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• 5 to 3 synthesis polymerization of DNA by
  condensing a 5-nucleoside triphophate (dNTP)
  onto the 3 OH gr. of another nucleotide
  (phosphodiester bond) by DNA pol III (dimer)
• Primase synthesizes a short RNA primer (3-10 nt.
  Long)
• New DNA synthesis is primed with a short segment
  of RNA
• DNA pol I 5-3 exonuclease, removal of RNA
  primer and replace them with dNTP using its DNA
  polymerase activity, DNA repair
• DNA ligase forming an intact new DNA strand

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Reaction catalyzed by DNA polymerase
Fig. 6.1
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Functions of Bacterial DNA polymerase
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(Proof reading)
5?3 Exonuclease
3 ? 5 Exonuclease
5-ATCGACGTTAAGGCCATCGTG-3 3-TAGCTGCAAT
TCCGGTAGCATAAGCATGATA-5
Endonuclease
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DNA replication Prokaryote

• Only one origin of replication (Ori)
• Bidirectional replication
• 5 to 3 DNA synthesis
• Leading strand
• Lagging strand or Okazaki fragment
• TER region Terminator Utilization Substance
  (TUS) binding ? termination of DNA replication
• Escherichia Coli (E.Coli)
• Circular chromosome

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5
Leading strand
3
3
5
Okazaki fragment
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Model of the E.coli replication fork
Fig. 6.10
Leading strand
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DNA synthesis in Eukaryotic cells

• Occurred in S phase
• Ori called Autonomously Replicating Sequences
  (ARS ) multiple ARS
• In human genome 30,000 Ori
• 10 times Slower than Prokaryote
• Okazaki fragment size
• Eukaryote 100-200 bp
• Prokaryote 1000-2000 bp

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ARS (yeast)
Fig. 6.15
ARS is the binding site for ORC (origin
recognition complex), AT rich seq.
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Functions of Eukaryotic DNA polymerase
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Origins of DNA replication on mammalian
chromosomes
origins of DNA replication (every 150 kb)
5 3
3 5
bidirectional replication
replication bubble
fusion of bubbles
5 3
3 5
daughter chromosomes
3 5
5 3
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Proliferating Cell Nuclear Antigen
RNaseH and 5-3 exonuclease, DNA pol d, ligase
replication factor C
replication protein A
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Accessory proteins

• RFC (replication factor C) is clamp-loading
  protein, which forms complex with PCNA that
  recognized and bind DNA at junction between
  primer and template.
• PCNA (proliferating cell nuclear antigen) is
  sliding clamp protein, which loads DNA pol. and
  maintains the association of the DNA pol. with
  DNA template

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• RPA is replication protein A stabilize the
  unwound template DNA. Keeping it as ssDNA

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DNA synthesis in Eukaryotic cells

• Telomerase Reverse transcriptase, carry its own
  template RNA, which complementary to the telomere
  repeat seq.
• Synthesis of telomeres without DNA template
• Restoration of ends end of lagging strand (RNA
  primer was removed)

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Telomerase Elongation of lagging strand template
Telomerase
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Action of telomerase
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Think about it!
5-ATCGCATGCGTTAACCGG-3
DNA template
3-TAGCGTACGCAATTGGCC-5
New DNA strand
Q ??????????????????????????????????
5'--ATCGATAG--3' ????????????????????????????????
??????????????????? 5'--?????--3 ?
A 5-CTATCGAT-3
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DNA repair
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Mutation

• Result from
• The incorporation of incorrect bases during DNA
  replication
• Exposure to various chemicals or radiation
• Damaged DNA can block replication or
  transcription and can result in a high frequency
  of mutation

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Proofreading activity of DNA polymerase

• 3-5 exonuclease activity selectively excise
  mismatched based at the end of growing chain.
• Reducing frequency error to one per to 108 and 109

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Proofreading of DNA polymerase
Fig. 6.11
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DNA repair

• Direct reversal of the chemical reaction
• Excision Repair
• Base-excision
• Nucleotide-excision
• Mismatch repair
• Translesion DNA synthesis
• Recombinational repair

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Example of DNA damage
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Alkylating agents ethyl methane sulfonate and
nitrosoguanidine
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Direct repair of Thymine dimer (UV light) by
photoactivation
photolyase
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Repair of O6-methylguanine
Figure 6.20
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Base-excision repair
Fig. 6.21
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Base-excision repair

• DNA glycosylase cleave bond btw base and sugar
• AP endonuclease (apyrimidinic or apurinic site)
  cleave phosphodiester bond adjacent to the AP site

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Nucleotide-excision repair
In E.coli
Thymine dimers
Uvr ABC endonuclease
Helicase
DNA polymerase Ligase
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Mismatch repair system
Old strand Methylated at GATC sequences
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Translestion DNA synthesis
SOS reponse ("Save Our Ship").
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Recombinational Repair
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Questions in the examinationII

• Prokaryotic DNA replication 5 points
• Eukaryotic DNA replication 5 points
• Telomerase activity 5 points
• DNA repair 5 points