Endonucleases
Endo = within · Nuclease = enzyme that cleaves DNA
- Restriction endonucleases
- DNases
- Homing endonucleases
- Cas9
The first two are this section. Homing endonucleases and Cas9 are endonucleases too, and they come later in the course.
Endonucleases cut DNA internally, in the middle of a sequence rather than in from an end. Because of that they work equally well on linear and circular DNA, which will not be true of anything in the next section. The class includes restriction endonucleases, homing endonucleases, DNases and RNases. Two of those are this section: the restriction endonucleases and DNase I. Homing endonucleases and Cas9 cut internally too and belong here by the same definition, but they are programmable nucleases and they get their own treatment later in the course.
Section divider: Endonucleases, defining the term and listing four kinds: restriction endonucleases, DNases, homing endonucleases and Cas9. A line below notes that the first two are covered in this section and that homing endonucleases and Cas9 come later in the course.
Restriction Endonucleases
- Type I
- RMS systems (3 subunit proteins)
- Typically named HsdR, HsdM, and HsdS
- Generally not sequence specific
- Not used in vitro; usually knocked out
- Type II
- Cleave at specific DNA sequences
- We’ll go through these in detail
- Type III
- Sequence-specific, but not in common use
- Type IV
- Cleave modified (methylated, etc.) DNA
- McrA, McrBC and Mrr, usually knocked out
A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, PMID: 12654995
Restriction endonucleases come in four types. Type I are the restriction-modification systems present in most prokaryotic genomes, hsdR, M and S in E. coli, and they do the native immune job of destroying incoming foreign DNA. They recognise some molecular pattern, but it is poorly defined and does not reduce to a simple sequence. You will never use one in vitro. They matter because knocking them out is necessary for efficient transformation. Type II are the ones you know, EcoRI and every other sequence-specific enzyme, and they are the rest of this section. Type III are sequence specific but not in common use. Type IV are like Type I in being endogenous and low specificity: they cleave methylated or otherwise modified DNA, are usually encoded by three proteins, and in E. coli that is McrA, McrBC and Mrr, also knocked out to get transformation efficiency up.
The four classes of restriction endonuclease as four headed groups: Type I, Type II, Type III and Type IV, each with its distinguishing properties.