SEMINAR 1: RESTRICTION ENDONUCLEASES
Nobel Prize 1978 to Werner Arber, Daniel Nathans and Hamilton O. Smith for the discovery
of restriction enzymes and their application to molecular genetics problems.
Restriction enzymes: dimers, two subunits (one for each chain of the double helix).
Each unit recognizes a string and size.
Enzymes used in recombinant DNA technology.
Restriction endonucleases: enzymes that have DNA or RNA as a substrate and that,
to be defined as such, have a specific affinity, a Vmax, certain activators…
Nucleases: break only phosphodiester bonds.
- Exonucleases: break the phosphodiester bonds in the strand from the outside,
either from 5 '→ 3' or 3 '→ 5', and degrade DNA.
- Endonucleases: they break the phosphodiester bonds from within the chain.
The cut is symmetrical and simultaneous in the two chains.
When cut, new ends are generated.
On the other hand, there are many enzymes that work with DNA in different ways:
RESTRICTION ENDONUCLEASES (TYPE II)
- Recognize specificsequences DNA(restriction targets) and cut the two strands, usually
within these sequences ( although they can do so outside of these) and
always by the same place (specificity of functionality).
- The substrate is DNA. They exclusively recognizeDNA double-stranded (double helix).
- Restriction targets are palindromic sequences shortof 4 (1/256) to 8 pairs
(sometimes odd) of bases (1/65536).
, - By breaking phosphodiester bonds, they release a phosphate at 5 'and a free hydroxyl at
3'.
- Of bacterial origin (prokaryotes), no eukaryotes present.
- They areenzymes homodimeric, since each subunit cuts one of the two strands of
DNA.
- Nomenclature:
- 3 first letters: refer to the organism from which they have
been isolated (the names of the restriction enzymes are
written in italics).
- Letter (may or may not be there): refers to the particular
isolated strain.
- Roman numeral: refers to the order in which this enzyme
was isolated.
Example: BamHI: Bacillus amyloliquefaciens, strain
(serotype) H, 1st enzyme obtained from this strain.
- Andsoeschizomers: different enzymes that recognize the same sequence and cut (or not) by
the same place.
- Neo-schizomers: different enzymes that recognize the same sequence but cut in different
places. Example: Sma I (GGG ^ CCC) vs. Xma I (G ^ GGCCC)
RESTRICTION-METHYLATION SYSTEM
- They are called "restriction enzymes" because they "restrict" the permanence of
bacteriophages. They are theimmune systembacterial "" that acts by destroying the
invading DNA to
protect itself.
- It can attack the same sequences of its own DNA. Not to mention that they are associated
with methylases.
Methylases: enzymes that recognize the same restriction sequence in bacterial DNA (host)
and methylene to protect it from digestion by its own restriction enzymes, which destroy
invasive bacteriophage DNA, which is not protected by methylation.
- There is one for each restriction enzyme (same specificity).
- Methylene in particular adenines.
Nobel Prize 1978 to Werner Arber, Daniel Nathans and Hamilton O. Smith for the discovery
of restriction enzymes and their application to molecular genetics problems.
Restriction enzymes: dimers, two subunits (one for each chain of the double helix).
Each unit recognizes a string and size.
Enzymes used in recombinant DNA technology.
Restriction endonucleases: enzymes that have DNA or RNA as a substrate and that,
to be defined as such, have a specific affinity, a Vmax, certain activators…
Nucleases: break only phosphodiester bonds.
- Exonucleases: break the phosphodiester bonds in the strand from the outside,
either from 5 '→ 3' or 3 '→ 5', and degrade DNA.
- Endonucleases: they break the phosphodiester bonds from within the chain.
The cut is symmetrical and simultaneous in the two chains.
When cut, new ends are generated.
On the other hand, there are many enzymes that work with DNA in different ways:
RESTRICTION ENDONUCLEASES (TYPE II)
- Recognize specificsequences DNA(restriction targets) and cut the two strands, usually
within these sequences ( although they can do so outside of these) and
always by the same place (specificity of functionality).
- The substrate is DNA. They exclusively recognizeDNA double-stranded (double helix).
- Restriction targets are palindromic sequences shortof 4 (1/256) to 8 pairs
(sometimes odd) of bases (1/65536).
, - By breaking phosphodiester bonds, they release a phosphate at 5 'and a free hydroxyl at
3'.
- Of bacterial origin (prokaryotes), no eukaryotes present.
- They areenzymes homodimeric, since each subunit cuts one of the two strands of
DNA.
- Nomenclature:
- 3 first letters: refer to the organism from which they have
been isolated (the names of the restriction enzymes are
written in italics).
- Letter (may or may not be there): refers to the particular
isolated strain.
- Roman numeral: refers to the order in which this enzyme
was isolated.
Example: BamHI: Bacillus amyloliquefaciens, strain
(serotype) H, 1st enzyme obtained from this strain.
- Andsoeschizomers: different enzymes that recognize the same sequence and cut (or not) by
the same place.
- Neo-schizomers: different enzymes that recognize the same sequence but cut in different
places. Example: Sma I (GGG ^ CCC) vs. Xma I (G ^ GGCCC)
RESTRICTION-METHYLATION SYSTEM
- They are called "restriction enzymes" because they "restrict" the permanence of
bacteriophages. They are theimmune systembacterial "" that acts by destroying the
invading DNA to
protect itself.
- It can attack the same sequences of its own DNA. Not to mention that they are associated
with methylases.
Methylases: enzymes that recognize the same restriction sequence in bacterial DNA (host)
and methylene to protect it from digestion by its own restriction enzymes, which destroy
invasive bacteriophage DNA, which is not protected by methylation.
- There is one for each restriction enzyme (same specificity).
- Methylene in particular adenines.
