notes

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. 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.

12. 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

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

14. 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”)

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

16. Unwinding the DNA by Helicase
(DnaB protein)
 Uses ATP to separate the DNA strands
 At least 4 helicases have been identified in
E. coli.
 NOTE: Mutation in such an essential gene
would be lethal.

17. 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’

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

19. 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

21. 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

22. 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

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

24. 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

25. 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

26. 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)

27. 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

28. 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

29. 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

30. 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

31. 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

32. 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.

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

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

35. 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

36. 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

37. 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 RNA se H
 DNA strand serves as template.
 Also catalyzed by RT
 Result:dsDNA
 New DNA integrates into host genome

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

39. 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

40. Eukaryotes
 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

42. • Requirements of replication:
• A template strand
• Raw material: nucleotides
• Enzymes and other proteins
Linear Eukaryotic Replication

43. • Direction of replication:
• DNA polymerase add nucleotides only to the
3′ end of a growing strand.
• The replication can only go 5′3′.
Linear Eukaryotic Replication

46. • Direction of replication:
• Leading strand: undergoes continuous
replication
• Lagging strand: undergoes discontinuous
replication
• Okazaki fragment: the discontinuously
synthesized short DNA fragments
forming the lagging strand
Linear Eukaryotic Replication

49. Eukaryotic DNA Replication
• Eukaryotic DNA polymerase
• DNA polymerase a- acts like Primase to initiate
• DNA polymerase d- replicates lagging strand
• DNA polymerase e- replicates leading strand

50. Eukaryotic DNA Replication
• Replication at the ends of chromosomes:
• Telomeres and telomerase

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