DNA modifying enzymes

Enzymes that modify DNA are useful because they

allow the investigator to manipulate DNA in defined
ways
- Polymerases elongate DNA molecules by adding
free nucleotides to the 3 ends (usually according to
an opposite template strand)
- Endonucleases cut DNA fragments in the middle
of the molecule
- Exonucleases degrade DNA from the ends
- Ligases join loose ends of DNA together

DNA polymerases
DNA polymerases exonuclease activities:
- Activity 35 exonuclease. ( proofreading activity)
allows the polymerase to correct errors by
removing a nucleotide that has been inserted
incorrectly.
- Activity 53 exonuclease activity is possessed by
some DNA polymerases.

Proof reading activity

of the 3 to 5 exonuclease.
DNAPI stalls if the incorrect
ntd is added - it cant add the
next ntd in the chain

Proof reading activity is slow

compared to polymerizing
activity, but the stalling of
DNAP I after insertion of an
incorrect base allows the
proofreading activity to
catch up with the polymerizing
activity and remove the
incorrect base.

The types of DNA polymerases used in research:

DNA polymerase I: Unmodified E. coli enzyme .
Use: DNA labeling .
Klenow polymerase: Modified version of E.coli
DNA polymerase I
Use: DNA labeling

The Enzymology
of DNA Replication

In 1957, Arthur Kornberg demonstrated the

existence of a DNA polymerase - DNA
polymerase I
DNA Polymerase I has THREE different
enzymatic activities in a single polypeptide:
a 5 to 3 DNA polymerizing activity
a 3 to 5 exonuclease activity
a 5 to 3 exonuclease activity

Functional domains in the Klenow Fragment (left) and DNA Polymerase I (right).

DNA polymerase I

Nick Translation

Nucleases

Exonucleases

Figure 1. Prepare single-stranded template with Lambda Exonuclease.

Figure. 1 Lambda
Exonuclease
selectively digests the
strand of a PCR
product
produced
using a PCR primer
with a 5-phosphate.
The resulting singlestranded PCR product
can be used for SSCP
analysis
or
sequencing.

Nucleases

Endonucleases

Endonucleases
I. Non specific
e.g. S1 nuclease, from the fungus
Aspergillus oryzae
And Deoxyribonuclease I
(DNaseI), from Escherichia coli

II. Specific
e.g. Restriction endonucleases,
from many sources

Endonucleases
I. Non specific
- S1 nuclease (Endonuclease specific for singlestranded DNA and RNA, from the fungus Aspergillus
oryzae

Use:Transcript mapping
- Deoxyribonuclease I (DNaseI) Endonuclease specific
for double stranded DNA and RNA, from Escherichia
coli

Use:Nuclease footprinting

S1 nuclease protection
digests only single-stranded RNA and DNA
Find introns:

exon 1

Digest with S1
Run gel

intron

exon 2 genomic DNA

antisense probe
exon 1
exon 2

Endonucleases
II. Specific
e.g. Restriction
endonucleases: Sequencespecific DNA endonucleases,
from many sources

Use:Many applications

Restriction endonuclease
Restriction Endonucleases
- Also called restriction enzymes
- Recognize, bind to, and cleave DNA molecules
at specific sequences (usually 4-6 base pairs in
length) but there are some that are 5, 8, or longer
- The double strand breaks can create ends that
are:
* Blunt, cutting both strands in the same place
* Sticky, with overhanging nucleotides on the 5
or 3 ends

Restriction endonuclease
Restriction enzymes
Over 10,000 bacteria species have been
screened for restriction enzymes
Over 2,500 restriction enzymes have been found
Over 250 distinct specificities
Occasionally enzymes with novel DNA sequence
specificities are still found while most now prove
to be duplicates (isoschizomers) of already
discovered specificities.

Restriction endonuclease
There are three types of restriction enzymes.
With Types I and III there is no strict control over the
position of the cut relative to the specific sequence in the
DNA molecule that is recognized by the enzyme. These
enzymes are therefore less useful .
Type II enzymes do not suffer from this disadvantage
because the cut is always at the same place, either within
the recognition sequence or very close to it

Type II Restriction enzymes are endonucleases that

cut DNA at specific sites, and are most useful for
molecular biology research

Restriction enzymes

Restriction enzymes are

molecular scissors

Restriction enzymes

Restriction Enzymes scan the DNA code

Find a very specific set of nucleotides
Make a specific cut

Restriction enzymes
Restriction enzymes recognize and make a cut
within
specific palindromic sequences, known as restriction sites,
in the genetic code. This is
usually a 4- or 6 base pair
sequence.

Picking a palindrome
Words that read the same forwards as backwards
hannaH
Hannah
Level

leveL

Madam

madaM

Restriction enzymes
Restriction Enzyme Recognition Sites
Restriction sites are general palindromic:
Able was I, ere, I saw Elba

5-GGATCC-3
Bam H1 site:
3-CCTAGG-5

HaeIII
HaeIII is a restriction enzyme that searches the
DNA molecule until it finds this sequence of
four nitrogen bases.

5 TGACGGGTTCGAGGCCAG 3
3 ACTGCCCAAGGTCCGGTC 5
5 TGACGGGTTCGAGGCCAG 3
3 ACTGCCCAAGGTCCGGTC 5

Once the recognition site was found

HaeIII could go to work cutting
(cleaving) the DNA
5 TGACGGGTTCGAGGCCAG 3
3 ACTGCCCAAGGTCCGGTC 5

These cuts produce what scientists call

blunt ends
5 TGACGGGTTCGAGG
3 ACTGCCCAAGGTCC

CCAG 3
GGTC 5

Restriction enzymes
Restriction enzymes are named based on the bacteria in
which they are isolated in the following example for the
enzyme EcoRI:
E
Escherichia (genus)
co coli (species)
R
RY13 (strain)
I
First identified Order ID'd in bacterium

Restriction enzymes
Nomenclature of Restriction Enzymes
The 1st letter (in capital and italics) = first initial of
Genus name (from which the enzyme was isolated
The 2nd and 3rd (in italics) = the first 2 letters of
the species name
e.g. Hin = Haemophilus influenzae
The 4th letter (sometimes in italics) = strain or type
e.g. Hind = Haemophilus influenzae Rd
The roman number followed is given to distinguish
different restriction and modification system in the
same strain
e.g. HindIII

EcoRI 5 G AATTC 3
3 CTTAA G 5

EcoRII

W=A or T

.....CCWGG
GGWCC.....

blunt ends and sticky ends

Remember how HaeIII produced a blunt end?
EcoRI, for instance, makes a staggered cut and
produces a sticky end
5 GAATTC 3
3 CTTAAG 5
5 GAATTC 3
3 CTTAAG 5
5 G
3 CTTAA

AATTC 3
G 5

Restriction enzymes
Single stranded nick

Eco RI Restriction Enzyme

Some more examples of restriction sites of

restriction enzymes with their cut sites:
HindIII: 5 AAGCTT 3
3 TTCGAA 5
BamHI: 5 GGATCC 3
3 CCTAGG 5
AluI: 5 AGCT 3
3 TCGA 5

Restriction Enzyme Recognition Sites

BglII

5 A-G-A-T-C-T
T-C-T-A-G-A 5

Sau3A

BamHI

5 G-A-T-C
C-T-A-G 5

All these sticky ends

are compatible

5 G-G-A-T-C-C
C-C-T-A-G-G 5

Isoschizomers: In certain cases, two or more different enzymes may

recognize identical sites. (e.g. MboI also cleaves at GATC, and so is an
isochizomer of Sau3A.)

Restriction enzymes
Frequency of cutting of recognition enzymes
Sau 3A (GATC) cuts ()()()() = once every 256 base
pairs (assuming G/C = A/T, which is often does not)
BamH1 (GGATCC) cuts ()()()()()() = once every
~4Kb
HindII (GTPyPuAC) cuts ()()()()()() = once
every ~1Kb
http://tools.neb.com/NEBcutter2/index.php

Ligation of compatible sticky ends

Corn DNA cleaved with EcoRI

Human DNA cleaved with EcoRI

5-C-G-G-T-A-C-T-A-G-OH
3-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4

PO4-A-A-T-T-C-A-G-C-T-A-C-G-3
HO-G-T-C-G-A-T-G-C-5

Complementary base pairing

5-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3
3-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5

+ DNA Ligase, + rATP

5-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3
3-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5

recombinant DNA molecule

Exercise1
HindIII 1/ 6160

EcoRI 5660

Eagl 542

PvuII 5116

YIP M
Apal 2035
PvuII 3547

SmaI 2860
SmaI 5 ccc ggg 3

How many base pairs in this plasmid?

How mamy fragments will be produced if this plasmid is digested with PvuII?

Agarose Gel Electrophoresis

_
DNA is negatively
charged from the
phosphate backbone
Agarose mesh

Visualize DNA with ethidium

bromide fluoresces orange
ONLY when bound to DNA

Gel Electrophoresis of DNA

What is Gel Electrophoresis?

Electro = flow of electricity, phoresis, from the Greek
= to carry across
A gel is a colloid, a suspension of tiny particles in a
medium, occurring in a solid form, like gelatin
Gel electrophoresis refers to the separation of charged
particles located in a gel when an electric current is
applied
Charged particles can include DNA, amino acids,
peptides, etc

Gel electrophoresis
Gel electrophoresis is a widely used technique for the
analysis of nucleic acids and proteins. Agarose gel
electrophoresis is routinely used for the preparation and
analysis of DNA.
Gel electrophoresis is a procedure that separates
molecules on the basis of their rate of movement through
a gel under the influence of an electrical field.

Why do gel electrophoresis?

When DNA is cut by restriction enzymes, the
result is a mix of pieces of DNA of different
lengths
It is useful to be able to separate the pieces - i.e.
for recovering particular pieces of DNA, for
forensic work or for sequencing

Gel with molecular weight marker

46

Summary
Restriction endonucleases recognize specific sequences in
DNA molecules and make cuts in both strands
This allows very specific cutting of DNAs 4-7
The cuts in the two strands are frequently staggered, so
restriction enzymes can create sticky ends that help to link
together 2 DNAs to form a recombinant DNA in vitro

Exercise

Plasmid vectors containing a polylinker

(a) Sequence of a polylinker that includes one copy of the recognition

site, indicated by brackets, for each of the 10 restriction enzymes
indicated. Polylinkers are chemically synthesized and then are inserted
into a plasmid vector. Only one strand is shown

1. The nucleotide sequence of a polylinker in a particular

plasmid vector is
GAATTCCCGGGGATCCTCTAGAGTCGACCTGCAGG
CATGCThis polylinker contains restriction sites for BamHI , EcoRI ,
PstI , SalI , SmaI , SphI , and XbaI . Indicate the location of
each restriction site in this sequence.
2. A vector has a polylinker containing restriction sites in
the following order: HindIII , SacI , XhoI , BglII , XbaI , and
ClaI .
- Give a possible nucleotide sequence for the polylinker .

Enzyme

Recognition
Sequence

BamHI

G GATCC

EcoRI

G AATTC

PstI

CTGCA G

SacI

GAGCT C

SalI

G TCGAC

SmaI

CCC GGG

SphI

GCATG C

XbaI

T CTAGA

XmaI

C CCGGG

Nucleases

What is difference between DNase and RNase?

DNase
RNases

cut DNA
cut RNA

RNases

Ribonuclease H (RNase H)

Replacement
Synthesis

DNA ligases
DNA fragments that have been generated by
treatment with a restriction endonuclease can be
joined back together again, or attached to a new
partner, by a DNA ligase. The reaction requires
energy, which is provided by adding either ATP or
NAD to the reaction mixture, depending on the
type of ligase that is being used.

DNA replication requires many

enzymes and protein factors
-

Replisome
Helicases
Topoisomerases
DNA-binding proteins
Primases
DNA ligases

DNA ligases

Application of DNA ligase

Role of Phosphatase in DNA ligation

Phosphotases & Kinases

Flow of Genetic Information :

The Central Dogma of Molecular Biology

DNA polymerase

Reverse
transcriptase

Alberts et al, 2002, p. 301

Reverse transcriptase
- An enzyme that catalyses the synthesis of a
DNA strand from an RNA template.
- The produced DNA called complementry
DNA (cDNA)
* Present in retro virus & other RNA viruses
* Application: Used in RT-PCR (e.g.
detection of HCV-Ag)

Reverse transcriptase