ENZYMES THAT MODIFY DNA AND RNA

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• ENZYMES THAT MODIFY DNA AND RNA
• 1. RESTRICTION ENDONUCLEASES AND METHYLASES
• 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

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• 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
• Require ATP for cleavage
• cleave DNA a substantial distance from the
  recognition sequence
• not commonly used
• Type II
• RE are not physically associated with methylases
• do not require ATP for cleavage
• generally cleave within or very near the
  recognition sequence
• isolated 100's of different type II REs, many of
  which are available commercially
• The first type II RE characterised was from
  E.coli and was designated E.coRI
• 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
• 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

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• NAMING R.E.
• A 3 letter abbreviation based on the genus and
  species of bacteria e.g. Eco E.coli
• a 4th letter can be used to indicate strain eg
  Hind
• Roman numerals are used to designate the order of
  characterisation of the different R.E. from the
  same organism
• e.g. HpaI and HpaII- the first and second R.E.
  isolated from Haemophilus parainfluenzae
• RECOGNITION SITES
• The palindromic sequences where most type II R.E.
  bind and cut a DNA molecule are called
  recognition sites
• Recognition sites of many type II R.E. contain
  4-6 specific nucleotides
• CLEAVAGE
• Can result in sticky ends or blunt ends

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• 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

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• Enzymes Practical considerations cont
• Manufacturers catalogues give optimum buffers,
  temps and stabilities
• If xs enzyme is used may result in non specific
  cutting (called star activity)
• Contamination of enzyme stocks is disasterous.
  Use clean tips all the time.
• Enzymes should be stored in -20C freezer(not
  frost free)
• Enzymes should be placed on ice immediately on
  removal from freezer
• Enzymes should be used immediately and then
  returned to freezer
• Diluted enzymes are generally unstable. Do not
  dilute for long term storage
• Wear gloves to prevent contaminating enzymes with
  proteases and RNases often present on fingers

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• 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

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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
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• 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)

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• 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

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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
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Polymerases cont.
4. Template independent polymerase terminal
deoxynucleotide transferase (TdT) No
template Useful for generating restriction sites
at blunt ends and labelling
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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 3'-gt5'Exonuclease Source and Properties
Taq No From Thermus aquaticus. Halflife at 95C is 1.6 hours.
Pfu Yes From Pyrococcus furiosus. Appears to have the lowest error rate of known thermophilic DNA polymerases.
Vent Yes From Thermococcus litoralis also known as Tli polymerase. Halflife at 95 C is approximately 7 hours.
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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.

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Kinase reactions
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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.

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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
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Ligase continued

• 2- Repair reaction
• H bonds are not enough to hold sticky ends
  together. A means of reforming the
  internucleotide linkage between 3OH and
  5phosphate groups is required and ligase does
  this

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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.

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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 RNADNA
  hybrids at sites of single base mismatches, and
  the cleavage products can be analyzed.