ENZYMES THAT MODIFY DNA AND RNA
1. RESTRICTION ENDONUCLEASES AND
METHYLASES
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RESTRICTION ENDONUCLEASES EXIST IN NATURE IN
PROKARYOTES
Prokaryotic cells have restriction modification systems and will
cleave foreign DNA that enters the bacteria cell (e.g.
bacteriophage) but not host DNA that has been protected or
modified by methylation
source of enzyme reagents, essential for generating recombinant
DNA molecules
Need to understand how they work in order to avoid problems
when manipulating recombinant DNA
TYPES OF RESTRICTION ENDONUCLEASES
There are 3 types; Type I, II and III
Types I and III contain the restriction and modification activities in the
same multiunit enzyme complex
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Require ATP for cleavage
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cleave DNA a substantial distance from the recognition sequence
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not commonly used
Type II
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RE are not physically associated with methylases
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do not require ATP for cleavage
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generally cleave within or very near the recognition sequence
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isolated 100's of different type II REs, many of which are available
commercially
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The first type II RE characterised was from E.coli and was
designated E.coRI
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EcoRI binds to DNA region with a specific palindromic sequence
of 6 bp and cuts between the G and the A residues on each strand
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It specifically cleaves the internucleotide bond between the
oxygen of the 3'C of the sugar of one nucleotide and the
phosphate group attached to the 5' carbon of the sugar of the
adjacent nucleotide
NAMING R.E.
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A 3 letter abbreviation based on the genus and species of bacteria
e.g. Eco = E.coli
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a 4th letter can be used to indicate strain eg Hind
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Roman numerals are used to designate the order of
characterisation of the different R.E. from the same organism
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e.g. HpaI and HpaII- the first and second R.E. isolated from
Haemophilus parainfluenzae
RECOGNITION SITES
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The palindromic sequences where most type II R.E. bind and cut
a DNA molecule are called recognition sites
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Recognition sites of many type II R.E. contain 4-6 specific
nucleotides
CLEAVAGE
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Can result in sticky ends or blunt ends
Enzymes; Practical considerations
• Expensive
• Many are cloned recombinant enzymes but still can be expensive
• One unit of a Restriction enzyme is defined as the amount that will
cut 1ug of a test DNA in 1h at optimum temp
• Rate of cutting is dependent on
1. Number of sites/ug DNA
2. linear, circular or supercoiled DNA
3. R.E. sites near ends may not cut well
4. Contaminated DNA may not cut well
5. More enzyme required if buffer conditions are not optimum
6. Ability to cleave depends on surrounding sequence
Enzymes; Practical considerations cont………
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Manufacturers catalogues give optimum buffers, temps and stabilities
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If xs enzyme is used may result in non specific cutting (called star activity)
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Contamination of enzyme stocks is disasterous. Use clean tips all the time.
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Enzymes should be stored in -20C freezer(not frost free)
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Enzymes should be placed on ice immediately on removal from freezer
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Enzymes should be used immediately and then returned to freezer
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Diluted enzymes are generally unstable. Do not dilute for long term storage
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Wear gloves to prevent contaminating enzymes with proteases and RNases
often present on fingers
ENZYMES THAT MODIFY DNA OR RNA cont.
2. Polymerases
The purpose of all polymerases is to join single nucleotides into a
polymer
5’ to 3’ polymerase activity
•All DNA polymerases use deoxynucleotide 5’ triphosphates (dNTPs)
•Removes 2 phosphate groups (releasing a pyrophosphate and using
the released energy) from NTP and attaches the newly exposed 5’
phosphate to the 3’ hydroxyl of another nucleotide, generating a
phosphodiester bond
•Most polymerases require a template
•Most require a primer
Polymerases can have other activities as well as polymerase
(building) activity:
3’ to 5’ exonuclease activity
Many polymerase have this activity, useful for proof reading
Removes single mismatches
Combination of 5’ to 3’ polymerase and 3’ to 5’ exonuclease activity is
particularly useful for making blunt ends and labeling 3’ ends
5’ to 3’ exonuclease activity
Only some polymerases have this activity
Useful for removing RNA templates for nick translation
Ribonuclease H activity
Present in a few polymerases
Degrades RNA in RNA/DNA complexes
Properties of polymerases
• Turnover number-nucleotides/min
• Processivity-how many nucleotides added before
disassociates
• Error frequency- how frequently generates a
mismatch(#errors/base pair)
• Errors are dependent on conditions, pH, conc dNTP,
divalent cations
• Every polymerase makes a mistake about 1 in
100000bp. Usually caught and proofread. The proofread
error frequency is 1/1000000, making an overall error
frequency of 1 in 10 billion bp)
Examples
1. DNA dependant DNA polymerase: E.coli polymerase 1
Acts primarily as proofreader (both 3 to 5 and 5 to 3 exonuc act and
polym act.)
Has RNase H act to degrade RNA primers
Plays role in replication
2. DNA dependant RNA polymerase: RNA polymerase
Transcribes ssRNA from dsDNA in transcription
Polymerase cont.
3. RNA dependant DNA polymerase: Reverse transcriptase
Makes DNA from RNA templates also has RNase H activity and can destroy
the RNA in an RNA DNA hybrid molecule
Polymerases cont.
4. Template independent polymerase: terminal deoxynucleotide transferase (TdT)
No template
Useful for generating restriction sites at blunt ends and labelling
Polymerases cont
Thermo tolerant polymerases used for PCR (polymerase chain
reactions) reactions
The total error rate of Taq polymerase has been variously reported
between 1 x 10-4 to 2 x 10-5 errors per base pair.
Pfu polymerase appears to have the lowest error rate at roughly 1.5 x 10-6
error per base pair
Vent is intermediate between Taq and Pfu.
Polymerase
Taq
Pfu
Vent
3'->5'
Exonuclease
Source and Properties
No
From Thermus aquaticus. Halflife at
95C is 1.6 hours.
Yes
From Pyrococcus furiosus. Appears to
have the lowest error rate of known
thermophilic DNA polymerases.
Yes
From Thermococcus litoralis; also
known as Tli polymerase. Halflife
at 95 C is approximately 7 hours.
Kinase
3. Kinase
– catalyses the transfer of the gamma phosphate group of ATP to the 5’
hydroxyl of polynucleotide (all phosphates have to be removed from end). By
combining a Phosphatase with a kinase the 5’ end of DNA can be labeled
with a labeled phosphate group.
• e.g. Polynucleotide Kinase
• It is a product of the T4 bacteriophage, and commercial preparations are
usually products of the cloned phage gene expressed in E. coli. The
enzymatic activity of PNK is utilized in two types of reactions:
• PNK transfers the gamma phosphate from ATP to the 5' end of a
polynucleotide (DNA or RNA). The target nucleotide is lacking a 5'
phosphate either because it has been dephorphorylated or has been
synthesized chemically.
• In the "exchange reaction", target DNA or RNA that has a 5' phosphate is
incubated with an excess of ADP - in this setting, PNK will first transfer the
phosphate from the nucleic acid onto an ADP, forming ATP and leaving a
dephosphorylated target. PNK will then perform a forward reaction and
transfer a phosphate from ATP onto the target nucleic acid.
Kinase reactions
Phosphatases
4. Phosphatase- catalyses the hydrolysis of 5’ phosphate groups from DNA
or RNA or single nucleotides. Often used to prevent relegation of plasmids
once they have been opened by restriction digest (since ligase requires a
5’ phosphate for ligation )
e.g. Alkaline phosphatase removes 5' phosphate groups from DNA and
RNA. It will also remove phosphates from nucleotides and proteins. These
enzymes are most active at alkaline pH
There are several sources of alkaline phosphatase that differ in how
easily they can be inactivated:
• Bacterial alkaline phosphatase (BAP) is the most active of the enzymes,
but also the most difficult to
• Calf intestinal alkaline phosphatase (CIP) most widely used in
molecular, less active than BAP, but it can be effectively destroyed by
protease digestion or heat
• Shrimp alkaline is readily destroyed by heat (65C for 15 minutes).
Primary uses for alkaline phosphatase in DNA manipulations:
• Removing 5' phosphates from plasmid and bacteriophage vectors
and preventing self-ligation
• Removing 5' phosphates from fragments of DNA prior to labeling
with labelled phosphate.
Ligase
5. DNA ligases catalyze formation of a phosphodiester bond between
the 5' phosphate of one strand of DNA and the 3' hydroxyl of the
another to permit joining of 2 DNA molecules together
• e.g. The most widely used DNA ligase is derived from the T4
bacteriophage. T4 DNA ligase requires ATP as a cofactor. It also requires
ds DNA.
• T4 RNA ligase can use ssRNA or ssDNA substrates
1- Ligation of DNA with
complementary cohesive
termini
Ligase continued
2- Repair reaction
• H bonds are not enough to hold sticky ends together. A means of
reforming the internucleotide linkage between 3’OH and
5’phosphate groups is required and ligase does this
Nucleases
6. Nucleases: DNase and RNase
Most of the time nucleases are evil when you are trying to preserve the
integrity of RNA or DNA samples.
Many types differing in substrate specificity, cofactor requirements, and
whether they cleave nucleic acids internally (endonucleases), chew in
from the ends (exonucleases) or attack in both of these modes.
The most widely used nucleases are DNase I and RNase A
Deoxyribonuclease I cleaves double-stranded or single stranded DNA.
• Cleavage preferentially occurs adjacent to pyrimidine (C or T) residues
• an endonuclease.
• Major products are 5'-phosphorylated di, tri and tetranucleotides.
• In the presence of magnesium ions, DNase I hydrolyzes each strand of
duplex DNA independently, generating random cleavages.
• In the presence of manganese ions, the enzyme cleaves both strands of
DNA at approximately the same site, producing blunt ends or fragments
with 1-2 base overhangs.
• DNase I does not cleave RNA
Some of the common applications of DNase I are:
• Eliminating DNA (e.g. plasmid) from preparations of RNA.
• Analyzing DNA-protein interactions via DNase footprinting.
• Nicking DNA prior to labeling by nick translation.
Nucleases cont.
Ribonuclease A is an endoribonuclease that cleaves
single-stranded RNA at the 3' end of pyrimidine residues.
• It degrades the RNA into 3'-phosphorylated
mononucleotides and oligonucleotides.
Some of the major use of RNase A are:
• Eliminating or reducing RNA contamination in
preparations of plasmid DNA.
• Mapping mutations in DNA or RNA by mismatch
cleavage. RNase will cleave the RNA in RNA:DNA
hybrids at sites of single base mismatches, and the
cleavage products can be analyzed.