Replication

1. REPLICATION

2. The Problem
 DNA is maintained in a compressed,
supercoiled state.
 BUT, basis of replication is the formation
of strands based on specific bases pairing
with their complementary bases.
  Before DNA can be replicated it must be
made accessible, i.e., it must be unwound

3. THREE HYPOTHESES FOR DNA REPLICATION
Models of Replication

4. (a) Hypothesis 1:
Semi-conservative
replication
(b) Hypothesis 2:
Conservative replication
Intermediate molecule
(c) Hypothesis 3:
Dispersive replication
MODELS OF DNA REPLICATION

5. PREDICTED
DENSITIES OF
NEWLY
REPLICATED
DNA
MOLECULES
ACCORDING
TO THE
THREE
HYPOTHESES
ABOUT DNA
REPLICATION

6. Meselson and Stahl
Conclusion: Semi-conservative replication of DNA

7. Replication as a process
 Double-stranded DNA unwinds.
The junction of the unwound
molecules is a replication fork.
A new strand is formed by pairing
complementary bases with the
old strand.
Two molecules are made.
Each has one new and one old
DNA strand.

8. Continuous synthesis
Discontinuous synthesis
DNA replication is semi-discontinuous

9. Features of DNA Replication
 DNA replication is semiconservative
 Each strand of template DNA is being copied.
 DNA replication is semidiscontinuous
 The leading strand copies continuously
 The lagging strand copies in segments (Okazaki
fragments) which must be joined
 DNA replication is bidirectional
 Bidirectional replication involves two replication
forks, which move in opposite directions

10. DNA Replication-Prokaryotes
 DNA replication is semiconservative.
the helix must be unwound.
 Most naturally occurring DNA is slightly
negatively supercoiled.
 Torsional strain must be released
 Replication induces positive supercoiling
 Torsional strain must be released,
again.
 SOLUTION: Topoisomerases

11. The Problem of Overwinding

12. Topoisomerase Type I
 Precedes replicating DNA
 Mechanism
 Makes a cut in one strand, passes other
strand through it. Seals gap.
 Result: induces positive supercoiling as
strands are separated, allowing
replication machinery to proceed.

13. Helicase
 Operates in replication
fork
 Separates strands to
allow DNA Pol to
function on single
strands.
Translocate along single
strain in 5’->3’ or 3’->
5’ direction by
hydrolyzing ATP

14. Gyrase--A Type II Topoisomerase
 Introduces negative supercoils
 Cuts both strands
 Section located away from actual cut is
then passed through cut site.

15. Initiation of Replication
 Replication initiated at specific sites:
Origin of Replication (ori)
 Two Types of initiation:
 De novo –Synthesis initiated with RNA
primers. Most common.
 Covalent extension—synthesis of new strand
as an extension of an old strand (“Rolling
Circle”)

16. De novo Initiation
 Binding to Ori
C by DnaA
protein
 Opens
Strands
 Replication
proceeds
bidirectionally

17. Unwinding the DNA by Helicase
(DnaB protein)
 Uses ATP to separate the DNA strands
 At least 4 helicases have been identified in
E. coli.
 How was DnaB identified as the helicase
necessary for replication?
 NOTE: Mutation in such an essential gene
would be lethal.
 Solution?
 Conditional mutants

18. Liebowitz Experiment
What would you
expect if the
substrates are
separated by
electrophoresis after
treatment with a
helicase?

19. Liebowitz Assay--Results
 What do these
results indicate?
 ALTHOUGH PRIMASE
(DnaG) AND SINGLE-
STRAND BINDING
PROTEIN (SSB) BOTH
STIMULATE DNA
HELICASE (DnaB),
NEITHER HAVE
HELICASE ACTIVITY
OF THEIR OWN

20. Single Stranded DNA Binding
Proteins (SSB)
 Maintain strand separation once helicase
separates strands
 Not only separate and protect ssDNA, also
stimulates binding by DNA pol (too much
SSB inhibits DNA synthesis)
 Strand growth proceeds 5’>>3’

21. Replication: The Overview
 Requirements:
 Deoxyribonucleotides
 DNA template
 DNA Polymerase
 5 DNA pols in E. coli
 5 DNA pols in mammals
 Primer
 Proofreading

22. A total of 5 different DNAPs have been reported
in E. coli
 DNAP I: functions in repair and replication
 DNAP II: functions in DNA repair (proven in 1999)
 DNAP III: principal DNA replication enzyme
 DNAP IV: functions in DNA repair (discovered in 1999)
 DNAP V: functions in DNA repair (discovered in 1999)
To date, a total of 14 different DNA polymerases
have been reported in eukaryotes
The DNA Polymerase Family

24. DNA pol I
 First DNA pol discovered.
 Proteolysis yields 2 chains
 Larger Chain (Klenow Fragment) 68 kd
C-terminal 2/3rd. 5’>>3’ polymerizing
activity
N-terminal 1/3rd. 3’>>5’ exonuclease
activity
 Smaller chain: 5’>>3 exonucleolytic
activity
nt removal 5’>>3’
Can remove >1 nt
Can remove deoxyribos or ribos

25. DNA pol I
 First DNA pol discovered.
 Proteolysis yields 2 chains
 Larger Chain (Klenow Fragment) 68 kd
C-terminal 2/3rd. 5’>>3’ polymerizing
activity
N-terminal 1/3rd. 3’>>5’ exonuclease
activity
 Smaller chain: 5’>>3 exonucleolytic
activity
nt removal 5’>>3’
Can remove >1 nt
Can remove deoxyribos or ribos

26. The structure of the
Klenow fragment of
DNAP I from E. coli

27. Requires 5’-3’ activity of DNA
pol I
Steps
1. At a nick (free 3’ OH) in the DNA
the DNA pol I binds and digests
nucleotides in a 5’-3’ direction
2. The DNA polymerase activity
synthesizes a new DNA strand
3. A nick remains as the DNA pol I
dissociates from the ds DNA.
4. The nick is closed via DNA ligase
Nick Translation
Source: Lehninger pg. 940

28.  5'-exonuclease activity, working together with
the polymerase, accomplishes "nick translation"
This activity is critical in primer removal
Nick Translation 2

29. DNA Polymerase I is great, but….
In 1969 John Cairns and Paula deLucia
-isolated a mutant bacterial strain with only 1%
DNAP I activity (polA)
- mutant was super sensitive to UV radiation
- but otherwise the mutant was fine i.e. it could
divide, so obviously it can replicate its
DNA
Conclusion:
 DNA pol I is NOT the principal replication
enzyme in E. coli

30. - DNAP I is too slow (600 dNTPs added/minute)
- DNAP I is only moderately processive
(processivity refers to the number of dNTPs
added to a growing DNA chain before the
enzyme dissociates from the template)
Conclusion:
 There must be additional DNA polymerases.
 Biochemists purified them from the polA
mutant
Other clues….

31. The major replicative polymerase in E. coli
 ~ 1,000 dNTPs added/sec
 It’s highly processive: >500,000 dNTPs
added before dissociating
 Accuracy:
 1 error in 107 dNTPs added,
 with proofreading final error rate of 1 in
1010 overall.
DNA Polymerase III

32. The 10 subunits of E. coli DNA polymerase III
Subunit Function
a
e
q
t
b
g
d
d’
c
y
5’ to 3’ polymerizing activity
3’ to 5’ exonuclease activity
a and e assembly (scaffold)
Assembly of holoenzyme on DNA
Sliding clamp = processivity factor
Clamp-loading complex
Clamp-loading complex
Clamp-loading complex
Clamp-loading complex
Clamp-loading complex
Core
enzyme
HoloenzymeDNA Polymerase III Holoenzyme (Replicase)

33. Activities of DNA Pol III
 ~900 kd
 Synthesizes both leading and lagging
strand
 Can only extend from a primer (either
RNA or DNA), not initiate
 5’>>3’ polymerizing activity
 3’>>5’ exonuclease activity
 NO 5’>>3’ exonuclease activity

34. Subsequent
hydrolysis of
PPi drives the
reaction
forward
Nucleotides are added at the 3'-end of the strand
The 5’ to 3’ DNA polymerizing activity

35. Leading and Lagging Strands
 REMEMBER: DNA polymerases require a
primer.
 Most living things use an RNA primer
 Leading strand (continuous): primer made
by RNA polymerase
 Lagging strand (discontinuous): Primer
made by Primase
 Priming occurs near replication fork, need to
unwind helix. SOLUTION: Helicase
 Primosome= Primase + Helicase

36. The Replisome
 DNA pol III extends on
both the leading and
lagging strand
 Growth stops when Pol
III encounters an RNA
primer (no 5’>>3’
exonuclease activity)
 Pol I then extends the
chain while removing
the primer (5’>>3’)
 Stops when nick is
sealed by ligase

37. Ligase
 Uses NAD+ or ATP for
coupled reaction
 3-step reaction:
 AMP is transferred to
Lysine residue on enzyme
 AMP transferred to open
5’ phosphate via
temporary pyrophosphate
(i.e., activation of the
phosphate in the nick)
 AMP released,
phosphodiester linkage
made
 NADNMN + AMP
 ATP ADP + PPi

38. DNA Replication Model
1. Relaxation of supercoiled
DNA.
2. Denaturation and untwisting
of the double helix.
3. Stabilization of the ssDNA in
the replication fork by SSBs.
4. Initiation of new DNA
strands.
5. Elongation of the new DNA
strands.
6. Joining of the Okazaki
fragments on the lagging
strand.

39. Termination of
Replication
 Occurs @ specific site opposite ori c
 ~350 kb
 Flanked by 6 nearly identical non-palindromic*,
23 bp terminator (ter) sites
 * Significance?
Tus Protein-arrests
replication fork
motion

40. FIDELITY OF REPLICATION
 Expect 1/103-4, get 1/108-10.
 Factors
 3’5’ exonuclease activity in DNA pols
 Use of “tagged” primers to initiate
synthesis
 Battery of repair enzymes
 Cells maintain balanced levels of dNTPs

41. Why Okazaki Frags?
 Or, why not 3’5’ synthesis?
 Possibly due to problems with proofreading.
 PROBLEM:
 Imagine a misincorporation with a 3’5’
polymerase
 How is it removed?
 How is the chain extended?
 Is there a problem after removing a mismatch?

42. Covalent Extension Methods
 Often called “Rolling
circle”
 Common in
bacteriophages
 NOTE: de novo
initiation of circular
DNA results in theta
structures,
sometimes callled
“theta replication”

43. Rolling Circle I
 Few rounds of theta-
replication
 Nick outer strand
 Extend 3’ end of outer
strand, displacing
original
 Synthesis of
complementary strand
using displaced strand
as template
 Concatamers cut by
RE’s, sealed
 Result several copies of
circular dsDNA

44. Rolling Circle I
 “Template “rolls”, extrudes leading strand
 Okazaki frags made on leading strand as it
emerges.

45. Rolling Circle I
NOTE: can get
single unit genomes
or multimeric copies

46. Rolling Circle II
 EX ΦX174
 Circular ssDNA chromosome
 Copy + strand using E. coli
replication proteins to make
ds circle (theta replication)
 Protein A (phage) cuts +
strand
 Rolling circle replication
 Protein A cuts at unit length
and circularizes (ligates)
released ss chromosome
 Replication continues

47. Reverse Transcription
 DNA replication in retroviruses
 RNA Dependent DNA polymerase
 Process:
 Retroviral RNA acts as template
 Primer—Segment of host cell t-RNA
 Result: DNA RNA hybrid
 RNA strand degraded by RNAse H
 DNA strand serves as template.
 Also catalyzed by RT
 Result:dsDNA
 New DNA integrates into host genome

48. cDNA
Library
 Made from
mRNA
 Steps
 1st strand
 RNAse H
 2nd strand
 Tailing
 Insertion
 Transform

49. Eukaryotic DNA Replication
 Much larger genomes with slower
polymerase
 Solution
 Multiple initiation sites
 More molecules of polymerase
 EX: DNA pola present in ~2-5 X105 copies/cell
 Histones an issue
 Still many questions

50. Completing the Ends of Non-circular
DNA
 THE PROBLEM?
 Solutions
 Phage T-7
 Eukaryotes

51. Phage Solution to Problem
 Phage DNA is linear
 Ends have repetitive complementary sequences
 After removal of 5’end RNA primer, are left with
a 3’ overhang
 Overhangs form H-bonds with complementary
overhangs, gaps filled in ligated. RESULT:
concatamers
 RE cuts concatamer into unit length genomes
with 5’ overhangs
 DNA pol extends 3’ ends, resulting in complete
unit length genomes.

52. Eukaryotes
 Solution: Telomeres
 At ends of chromosomesare non-coding
regions, >1000 tandem repeats of GC rich
sequence.
 Telomeric DNA synthesized and
maintained by Telomerase
 Adds tandem repeats of TTGGG
 Is a ribonucleoprotein, uses internal
ribonucleotide sequences as a template

53. Telomeres
 Elongation
 Translocation
 Elongation
 New primer
synthesis
 Dna
Replication
 Primer removal
 Repeat