DNA Synthesis

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DNA Synthesis
Lecture 31
Mukund Modak, Ph.D.
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• DNA Objectives
• Define a DNA polymerase reaction and its
  components.
• Where does DNA replication begin? Where does it
  terminate?
• Know about leading and lagging strand synthesis.
• How many primers are required for each strand
  synthesis?
• What are Okazaki fragments? Remember some of the
  components required in lagging strand synthesis
  such as Pol I to remove RNA primers and DNA
  ligase to join the Okazaki fragments.
• Know the polarity of DNA to be replicated.
• Know the major eukaryotic DNA polymerases.
• Remember clamp protein( PCNA in eukaryotes and B
  clamp in prokaryotes) that clamps DNA pol (
  mainly delta and epsilon) to template and speeds
  up polymerase rate.
• What does enzyme telomerase do? It has RNA
  component with sequence complimentary to
  telomeric( DNA) sequences.

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Watson-Crick Model
Base

• Double helix
• Antiparallel
• Base-paired
• Base stacking
• Major groove
• Minor groove
• Sugar-phosphate
• Backbone
• Helix structure by X-ray diffraction

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2
O
P
Base
O
5
O
P
Base
Postulations Semi conser- vative replication via
base pairing principle. Expression via
transcription (by same base pairing rule)
O
5
3
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Review of Basics

• DNA replication is semiconservative

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Review of Basics

• DNA is synthesized by the addition of a
  deoxynucleotide to the 3? end of a polynucleotide
  chain
• Base-pairing between incoming dNTP and template
  strand provides specificity

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Antiparallel Double Strand
5
3
5
3
Base
Polymerization reaction
O
5
OH 5 PPP 3O - P - 5O PPi
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P-P-P-O-H2C
Therefore chain extends 3 to 5 Overall
growth 5 to 3
3
OH
Base
O
5
5
3
P-P-P-O-H2C
OH
5P
New chain
3
5P
OH
5
3
OH
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DNA dependent DNA polymerases are key enzymes for
DNA synthesis Requirements Template, primer,
4dNTPs, Mg 2
? Nucleotidyl transferase reaction ? Template
dependent dNTP selection ? direction of
synthesis 5 ? 3
Imp Primer (DNA or RNA) with 3OH, a minimum of
10 bases hydrogen bonded to template
5
3
3OH
3OH
3OH
5
5
5
Enzyme
Mg.dNTP
Primer n1 PPi
Prokaryotes contain 3 DNA polymerases I, II and
III
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Specialized enzymes and factors

• Specialized polymerases that synthesize primers
  and DNA
• Editing exonucleases to work with polymerases.
• Topoisomerases that convert supercoiled DNA to
  relaxed form
• Helicases that separate two parental strands of
  DNA
• Accessory proteins promoting tight binding of
  polymerase to DNA and
• thereby increase the speed of polymerases
  (sliding clamps)
• Details of the process

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Leading vs Lagging Strand Replication
3
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DNA REPLICATION PROTEINS
dnaA Ori C dnaB begins unwinding
(helicase) Rep Helicase Ongoing
unwinding SSB Stabilize ss DNA Gyrase
and Topoisomerase Supercoil to relax
transition Primase Synthesis of primer
(RNA) Pol III complex DNA synthesis Pol
I Primer removal and gap filling DNA
ligase Join DNA ends Topo IV Decatenates
replicated circles
Topo IV
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REPLICATION FORK SUMMARY
Begins with unwinding of two strands with
opposite polarity Two replisome assemblies
(Primase Pol III complex) Leading strand
(continuous synthesis) Lagging strand
(discontinuous synthesis) a. Looping of the
strand to transiently change polarity and
permit primer synthesis b. DNA synthesis in
pieces (Okazaki fragments) c. Pol I to remove
RNA primers and replace by DNA d. DNA ligase to
join small DNAs into a single large molecule
with ATP or NAD as a cofactor Termination
signal TUS factor Two forks reach each other at
mid point of the circle and are stalled by TUS
factor Replication completes with generation of
catenated circles. Seperation of circles by Topo
IV
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PROOF READING ACTIVITY OF POL I (3 to 5
exonuclease activity)
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Structure of first DNA polymerase (Klenow
Fragment of E.coli pol I, 1985)
Nomenclature of various structural units
(1992) Thumb Palm Fingers 3 5 exonuclease
The crystal structures of numerous DNA
polymerases solved later, showed same general
anatomy
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Lecture 8
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Eukaryotic DNA replication
G0, G1, S and M Phases (DNA replication in S
phase)
Many polymerases and accessory factors required
Multiple initiation points to replicate (3
billion bps) Linear chromosome Overall
replication scheme similar to prokaryotes Problem
with 5 RNA primer removal and fill
up Solution to this problem is telomerase action
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Eukaryotic DNA Polymerases

• a Repair and Replication and primase function
• Repair function
• g Mitochondrial DNA polymerase
• d Replication with PCNA (processivity factor)
• Replication
• ? Repair function
• i Repair function

Telomerases
Terminal deoxynucleotidyl transferase Viral
reverse transcriptase Viral replication
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Eukaryotic Fork

• Three polymerases required for replication
• Pol? synthesizes primer RNA and very little DNA
  (non-processive, no proofreading exonuclease)
• Pol? and Pol? both are processive (interact with
  PCNA) and have proofreading exonucleases

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Important Participants In Eukaryotic DNA
Replication

• Pol (alpha) (delta) and (epsilon) primer
  synthesis and DNA synthesis (There is a separate
  primase in prokaryores)
• Sliding clamp or processivity factor PCNA
  Increases rates and length of DNA (Equivalent to
  ß clamp in prokaryotes)
• FEN-1 nuclease removes RNA primers (In
  prokaryotes, 5- nuclease activity of pol I does
  it)
• RPA is a single strand binding protein (SSB in
  prokaryotes)

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End-Replication Problem
5 3

5 3
Process Okazaki Fragments
5 3

5 3
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Telomere Structure
5
3
G-rich
C-rich

• Telomeres composed of short (6-10 bp) repeats
  (2000 3000)
• G-rich in one strand, C-rich in other
• TTAGGG/CCCTAA

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TERMINAL DEOXYRIBONUCLEOTIDYL TRANSFERASE (TdT)
5
3
(ds DNA) Or (ss DNA)
TdT
5
3
DNAn1 PPi
dNTP Mg2
5
3

• Addition of dNTPs on to 3 OH end of DNA
• No template requirement
• No template guidance but will extend 3OH ends of
  ss or ds DNA
• with all 4 dNTPs

Biological novelty

• Found in only vertebrate species
• In preimmunocytes (thymus)
• Absent in mature circulating lymphocytes
• Used as a lympho blastic leukemia marker
• Involved in generation of diversified antibody
  genes

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AZTTP as a Chain Terminator