Biochemistry I, Spring Term
Lecture 36
April 22, 2013
Lecture 36: Restriction Enzymes, Ligase, DNA Polymerases, PCR
Overall Goal: To produce HIV protease from a drug resistant HIV virus. The protein will be made in E. coli (a
widely used bacteria) using recombinant DNA methods. The structure of the purified protein can be determined
and new HIV protease inhibitors can be designed that will be effective against the altered virus. The overall
procedure is as follows:
1) Isolate the genetic material (RNA) from the HIV virus that encodes the HIV protease.
2) Convert this
material to DNA
using polymerases,
amplify HIV protease
coding region using
PCR (polymerase
chain reaction)
2
1
vRNA
expression
vector
(plasmid)
DNA coding
HIV protease
3
3) Insert the DNA
encoding HIV protease into
Leader peptide
a bacterial expression vector (a
e
Stop codon
Start Codon
specialized form of a DNA
g
Ribosome binding
f HIV Protease
site
Coding Region
plasmid) using restriction
mRNA
d
Termination
Lac Operator
d
endonucleases and DNA ligase.
c
Promoter
Verify sequence using DNA
sequencing.
Properties of the expression vector:
a
b
a) Provide antibiotic resistance to the
Antibiotic
Resistance
host bacteria, such that only bacteria
Origin of
Gene
with the plasmid will grow in the
Replication
presence of the antibiotic (selective
4
pressure for maintaining the plasmid)
b) An origin of DNA replication so that the
Plasmids
plasmid will be replicated with the
[HIV protease expression
Chromosomal
vector] lac operator
DNA
bacterial DNA
Cell wall
c) DNA sequences that cause the production
o
o
o
o
o
Cell
of mRNA, copying the information in the
membrane
o
lacI
DNA to mRNA, including a regulated on/off
o
switch (lac operator).
o
o
o
o
d) Sequences in the mRNA that start and stop
Ribosomes
the production of the recombinant protein.
e) Coding region for protein to be expressed
5
(HIV protease).
f) Sequences in the mRNA that cause the
recombinant protein to be exported out of the
Cell
Number
cell, to facilitate purification (leader peptide).
(------)
g) His6 tag for affinity purification.
HIV
4) Transformation of the bacteria with the plasmid.
Protease
6
(- - - - )
5) Growth of the transformed bacteria.
7
6) Initiation, or induction, of the production of the
time
Affinity
recombinant protein.
chromatography
8
7) Purification of the recombinant protein.
Ala82
8) Structural and functional studies.
Asp25'
1
Asp25
Biochemistry I, Spring Term
Lecture 36
April 22, 2013
DNA Modifying Enzymes:
A. Restriction Endonuclease: [endo
- cut within, nuclease - cleave nucleic
acid]. Used by bacteria to degrade
invading viral DNA. Named after
bacterial species the particular
enzyme was isolated from, i.e. Eco =
E. Coli.
1. Enzyme binds to specific
recognition sequences with near
absolute specificity and high
affinity (KD = 10-10 M).
2. Enzymes usually bind in major
groove, forming both specific and
non-specific interactions.
3. Homodimeric enzymes have 180 degree rotational symmetry. Because
of the symmetry in the enzyme, the DNA sequence also symmetrical.
The sequence is the same on the top and bottom strands (referred to as
palindromic sequences).
4. Require Mg2+ for cleavage, usually cleave both strands at the same
position.
Enzyme
Recognition
Sequence
EcoRI(G^AATTC):
-G-A-A-T-T-C
-C-T-T-A-A-G
-G
-C-T-T-A-A
PvuI(CGAT^CG):
-C-G-A-T-C-G-G-C-T-A-G-C-
-C-G-A-T
-G-C
EcoRV(GAT^ATC):
-G-A-T-A-T-C-C-T-A-T-A-G-
-G-A-T
-C-T-A
HaeIII (GG^CC):
-G-G-C-C
-C-C-G-G
O
O
O
G
O
O
O P
O
O
O
OH
O
O P
O
O
A
O
A
O
O
Products
A-A-T-T-CG-
5’ overhang = sticky end
C-GT-A-G-C-
3’ overhang = Sticky end
-G-G
-C-C
A-T-CT-A-G-
blunt end
C-CG-G-
blunt end
Example: EcoR1: G^AATTC
a) Non-specific interactions with DNA phosphates.
---G-A-A-T-T-C-----C-T-T-A-A-G-----G A-A-T-T-C-----C-T-T-A-A G-----G
---C-T-T-A-A
A-A-T-T-C--G---
b) Specific hydrogen bonds with donor and acceptors at the edge of bases in the major groove:
Arg145
G
NH2
Asn141
C
N
O
A
N
H
T O
O
N
O
T
C
H
H3C
A
2
G
N
H
H
N
T
N
N
O
A
N
N
O
T
N
O
O
A
O
minor groove
G
Biochemistry I, Spring Term
Lecture 36
April 22, 2013
B. DNA Ligase – Uses ATP to join 5’phosphate to 3’-OH, provided the two groups are
held in close proximity. Complementary overhangs and blunt ends can be ligated.
Fragments created by the same restriction enzyme can always be joined to each other.
Sticky end ligation:
-G
A-A-T-T-C-
-G A-A-T-T-C-
--G-A-A-T-T-C--
G-
-C-T-T-A-A G-
--C-T-T-A-A-G—
-C-T-T-A-A
O
O
G
G
O
O
ADP + Pi
ATP
OH
O
O P
O
O
O
O P
O
O
O
O
A
A
O
O
Blunt end ligation:
-G-A-T
-C-T-A
A-T-CT-A-G-
-G-A-T A-T-C-C-T-A T-A-G-
-G-A-T-A-T-C-C-T-A-T-A-G-
C. DNA Polymerases: DNA polymerases play an important role in the replication of DNA.
5'-3'
3'-5'
Polymerases
Template Monomers
Polymerase Exonuclease
DNA Pol III (E. coli)
DNA Pol I (E. coli)
Reverse Transcriptase (HIV)
DNA
DNA
RNA/DNA
dNTP
dNTP
dNTP
1. Utilized a template to direct the order of added bases.
2. DNA polymerases require a basepaired primer with a 3’OH, RNA poly. do not.
3. Synthesize chains from 5'3' direction. Adding new bases to the 3' hydroxyl of
the existing polymer.
4. Use nucleotside triphosphates, producing a chain one base longer +
pyrophosphate (PPi). The PPi is hydrolyzed to insure a large negative
G for the reaction. Yet another example of indirect coupling in
O
biochemical reactions!
N
N
H
ribose
5. Fidelity of base incorporation is dependent on Watson-Crick baseO
pairing (A-T, G-C), plus purine-pyrimidine matching (see right).
T
Phosphodiester bond formation occurs quickly (1 msec) when the
H
correct match is made. Bond formation is slow when the bases are
N H
N
incorrectly matched – allowing time for proofreading. An example of
N
N
kinetic control of substrate specificity.
ribose
N
6. Incorrectly incorporated bases are removed by a proofreading
A
activity: 3'5' exonuclease activity.
H
i) This activity is found in most polymerases.
N H
N
ii) This activity is absent in many viral polymerase (e.g HIV rev.
N
N
H
ribose
trans.), leading to high mutation rates in the virus and a high
N
A
number of viruses that are resistant to HIV drugs.
O
H
N
N
O
ribose
T
O
H N
N
T
O
N
N
N
N
H
G
O
O
O
O
O
O
P
O
O
HO
O
P
O
O
3
P O
O
O
O
P O
O
O
O
O
OH
O
P O
O O
OH
O
T
C
O
O
P O
O
O
O
P O
O O
O
O
O
O
P O
P O
O
O
P
O
O
G
P O
O
O
O
O
P
O
O
O
O
O
O
H2O
O
O
T
O
P
O
P
O
O
C
O
O
O
O
G
T
O
O
O
A
O
P
O
O
O
O
O
C
H
O
P
O
P
Go<<0
O
P
ribose
N
O
O
ribose
O
O
O
Biochemistry I, Spring Term
Lecture 36
5'3' Polymerase:
5'
G-C-T3'OH
3'
C-G-A-T-A-C-C
April 22, 2013
G-C-T-A3'OH
C-G-A-T-A-C-C
G-C-T-A-T3'OH
C-G-A-T-A-C-C
3'5' Exonuclease:
5'
3'
G-C-T-T3'OH
C-G-A-T-A-C-C
G-C-T3'OH
C-G-A-T-A-C-C
Generation of dsDNA from HIV viral RNA (Step 1):
Reverse transcriptase has the following activities:
i. RNA dependent DNA polymerase activity.
ii. RNase activity, degrades RNA template.
iii. DNA dependent DNA polymerase activity.
expression
vector
(plasmid)
1
vRNA
cDNA
DNA coding
HIV protease
2
3
Leader peptide
Start Codon
Ribosome binding
site
Lac Operator
Promoter
e
f
HIV Protease
Coding Region
d
c
a
Antibiotic
Resistance
Gene
1A. Generate 1st DNA strand
DNA primer I
vRNA
(10 kb)
partially digested vRNA
cDNA
(copy DNA)
1B. Generate 2nd DNA strand.
DNA primer II
~600 bp
dsDNA
HIV protease
coding sequence
4
g
d
b
Origin of
Replication
Stop codon
mRNA
Termination
Biochemistry I, Spring Term
Lecture 36
April 22, 2013
PCR = Polymerase Chain Reaction (Step 2):

PCR is a method of amplifying a user-selected
segment of DNA within a larger DNA molecule.
 The location of the amplified segment is defined by
two primers, they anneal to their templates
according to W-C basepairing rules and initiate
polymerization from those sites.
 The final product contains the DNA between the
two primers and the primers themselves.
 Any non-complementary bases at the 5’ end of the
primers are also incorporated into the PCR product.
The actual reaction mix contains:
i. Double stranded DNA template
ii. Excess “right” and “left” primers
iii. dNTPs
iv. Thermostable DNA polymerase
The last item on the above list is quite important, since
each cycle of PCR requires a heating step to denature
the newly synthesized DNA and to allow the primers
to bind. Therefore the polymerase must be able to
withstand high temperature.
The amplification cycle [DNA amplification = DAP]
consists of the following three steps, repeated 15-25
times. After the third cycle, each cycle doubles the
amount of PCR product.
i. Denaturation of double stranded DNA (T=95 C)
[Denature]
ii. Annealing of primers on each strand (T=55 C)
[Anneal]
iii. Primer extension with DNA Polymerase + dNTPs (T ~ 78 C) [Polymerize]
The final PCR product encoding the HIV protease gene is:
5' CGGAATTCCCTCAGATCACTCTTTGGCAA.......TTTAAATTTCGGATCCCG 3'
3' GCCTTAAGGGAGTCTAGTGAGAAACCGTT.......AAATTTAAAGCCATGGGC 5'
EcoR1 ProGlnIleThrLeuTrpGln--------LeuAsnPhe BamH1
The sequences at each end, containing GAATTC and GGATCC are not part of the HIV
genetic material. These DNA sequences were added during the PCR reaction and contain
restriction endonuclease sites that will be used to precisely insert the HIV gene into the
expression plasmid.
C-G-G-A-A-T-T-C-HIV--G-G-A-T-C-C-C-G
G-C-C-T-T-A-A-G-Prot-C-C-T-A-G-G-G-C
C-G-G
G-C-C-T-T-A-A
Primer Design:
1. Sufficiently long that it will hybridize (bind via
Watson-Crick H-bonds) uniquely to the desired location.
2. Sufficiently long that it will hybridize during the
annealing step, e.g. TM = 60 C if the annealing T is 55C.
TM .= 81.5 +0.41(%GC) - 675/N
(675/N accounts for the dependence of melting
temperature on length):
3. May contain additional sequences at the 5’ ends of the
primers that will result in the incorporation of restriction
site, plus additional bases (two in this example) to
facilitate binding of the res. endonuclease to the DNA.
5
A-A-T-T-C-HIV—-G
G-Prot-C-C-T-A-G
G-A-T-C-C-C-G
G-G-C
Biochemistry I, Spring Term
Lecture 36
April 22, 2013
We would design primers that are the sequence of the start (top strand) and end (bottom strand) of the HIV
protease DNA coding region, with an EcoR1 site (GAATCC) on the left primer and a BamHI site (GGATCC) on
the right primer. The EcoR1-containing left primer would prime DNA synthesis using the lower strand as the
template and the BamH1-containing right primer would prime synthesis using the upper strand as the template.
5'CGGAATTC
CCTCAGATCACTCTTTGGC3’
5'--------TTCCCTCAGATCACTCTTTGGCAA-------TACTTTAAATTTCCCCATTAGTCCTATT-----3'[U]
ProGlnIleThrLeuTrpGln-----------LeuAsnPhe
3'--------AAGGGAGTCTAGTGAGAAACCGTT-------ATGAAATTTAAAGGGGTAATCAGGATAA-----5'[L]
3’TGAAATTTAAAG
CCTAGGGC5'
The final product, after PCR.
5' CGGAATTCCCTCAGATCACTCTTTGGCAA.......TTTAAATTTCGGATCCCG 3'
3' GCCTTAAGGGAGTCTAGTGAGAAACCGTT.......AAATTTAAAGCCATGGGC 5'
EcoR1 ProGlnIleThrLeuTrpGln--------LeuAsnPhe BamH1
HIV prot
coding region
HIV genome 1
10,000
Cycle 1
5'
3'
C 5'
G
G
A
A
3'
T
T
CCCTCAGATCACTCTTGGC
3'
5'
Denature
5'
3'
5'
3'
Anneal
3'
5'
3'
3'
5'
5'
Polymerize
5'
3'
3'
5'
5'
3'
5'
3'
Cycle 2 5'
Cycle 3
5'
3'
3'
5'
3'
5'
5'
Denature
Denature
5'
3'
5'
3'
5'
Anneal
5'
Anneal
5'
3'
5'
5'
Polymerize
Polymerize
5'
5'
3'
5'
6
5'