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enzymes (nucleases
enzyme that cleaves nucleic acids into smaller chains…
enzymes
nucleases
- enzyme that cleaves nucleic acids into smaller chains of nucleotides
2 mechanisms to break phosphodiester (P-O) bonds:
- Hydrolysis (most common)
- Lyase
- both methods leave 5’ P & 3’ OH
damage, structure, or sequence specific:
- damage: recognise the presence of any DNA then bind and scan for damage
- structure: recognise the intermediates generated by DNA repair mechanisms
- sequence: recognise a particular sequence of DNA
substrate specificity:
- DNA only: deoxyribonuclease
- RNA only: ribonuclease
- or both DNA & RNA
approach:
- Exonuclease: hydrolyses from either the 5’ or
3’ end of either ss- or dsDNA
~ 5’ exonucleases
~ 3’ exonucleases
~ or non-specific exonucleases
- Endonuclease: hydrolyse an internal P-O bond
DNase I
- to remove DNA from a solution
- cleaves ss- or dsDNA
- endonuclease (cleaves near C or T bases)
- cleavage results in di-, tri- and tetranucleotides
- acts differently depending on which cofactor is present
- If Mg2+ is present
~ cuts each strand independently
~ leaves random fragments
- If Mn2+ is present
~ Cleaves each strand close to
each other
~ Leaves dsDNA with 1 to 2 nt overhangs (sticky ends)
Exonuclease III
- preferentially remove dsDNA from a solution
- Removes mononucleotides from the 3’ termini of dsDNA
- Prefers blunt or 5’ overhangs
- Will act on 3’ overhang or nicks
- Inactive on ssDNA
Nuclease S1
- to remove ss overhangs to leave blunt ends
- Exonuclease
- Specificity depends on concentration
~ Low concentration = cuts both ss- & dsDNA
~ High concentration = cuts only dsDNA, dsRNA or DNA:RNA
Restriction Endonucleases
- Sequence specific
- Cut the DNA in a repeatable way into pieces of varying length
- Many hundreds known
- Used in cloning to enable DNA from two different sources to be prepared for joining
- Involved in Host Restriction- Modification systems
polymerases
- Catalyse the addition of a dNTP to the 3’ end of a DNA or RNA primer, the identity of the dNTP is dependent on the template
DNA polymerase I
- Has three different catalytic activities
~ 5’ to 3’ polymerase
~ 5’ to 3’ exonuclease
~ 3’ to 5’ exonuclease
Klenow
- Add bases to the 3’ end of DNA fragments to
fill in overhangs
- Remove 3’ overhangs to create blunt ends
- 5’ to 3’ exo can be used to incorporate labelled dNTPs into a strand
~ Creates labelled probes/ primers
- It will displace downstream bases as it extends
T4/ T7 DNA polymerases
- Same basic properties as Klenow, but won’t
displace downstream strand
- T4: 3’ to 5’ exonuclease is 200x more active than Klenow or T7
- T7: higher processivity than T4 or Klenow
Terminal Deoxynucleotidyl Transferase
- Doesn’t require a template (template
Independent)
- Can use either ds or ss DNA but must be greater than 3 bp long
- Adds bases to the 3’ end of DNA
- Prefers 3’ overhangs
- Blunt is also possible
- Requires Mg2+ and dNTP
- Used to create overhangs for cloning
Thermostable DNA polymerases
- Resistant to heat denaturation
- Sourced from thermophilic bacteria
- Polymerase activity maximal at 60- 75°C
bacterial restriction modification systems
- Primitive immune system found in bacteria and other prokaryotes, and provides defence against foreign DNA (such as that from bacteriophages)
- source of Restriction Endonucleases
- An RE (restriction enzyme) combines with a methylase (modification)
- Key characteristic: each pair of enzymes recognises a unique sequence in DNA
Restriction Endonuclease (RE)
- aka restriction enzyme
- Endonuclease that cuts dsDNA
- Will cut only at the site specified by its recognition capacity
Methylase (M)
- Adds a methyl group to DNA
- Does not affect base-pairing
- Adds Methyl group only at the site specified by its recognition capacity
RMS - RE + M
- RE acts as a frontline defence for the cell by digesting any invading DNA
~ But, this is not a good system because it would cut its own DNA as well
- but, if the recognition site for an RE is methylated,
~ RE cannot cut the DNA
~ The “self” DNA is protected from cleavage
~ Invading DNA is not methylated so will be cleaved
- Drawback: If the first enzyme that the viral DNA encounters is M, then it gets protected
~ Cells usually have 2 or more independent RMS to overcome this problem
- not inheritable
- only maintained if the virus replicates inside
that cell
- Any replication outside that cell will lead to removal of the methyl group by dilution during replication
- RMS are not essential for cell survival
~ Except in the event of viral attack
- 3 types of RMS
~ Types I, II (common) & III
~ Differences are in the type of recognition sequence
ligases
- join Okazaki fragments during DNA replication
- catalyse the formation of P-O bonds between the 5’ P and 3’ OH of two strands
- can join strands with compatible overhangs, or blunt ended strands
- differ in minor characteristics
~ Thermal stability
~ Substrate specificity
- All will seal nicks in dsDNA
- They DO NOT add new bases
- Most common is T4 DNA ligase (from T4 bacteriophage)
~ requires ATP as a cofactor
- Also E. coli DNA ligase, Taq DNA ligase