2. 2
What are restriction enzymes?
●
Molecular scissors that cuts DNA.
●
Identifies specific Recognition sites.
●
Found naturally in prokaryotes as a defence
mechanism.
●
Do not cut host DNA- But how?
●
A useful tool in DNA modification and
manipulation.
3. 3
BRIEF HISTORY
Year Significant Event Scientists Involved
1950s Host controlled restriction
and modification
Luria, Weigle and Bertani
1960s
Restriction caused by
enzymatic cleavage of
phage DNA
Arber and Meselson
1970
Type II restriction
enzyme(RE) discovered
Smith, Kelly and Wilcox
1970s Use in DNA Mapping Nathans and Danna
1978 Nobel Prize for Physiology
or Medicine
Werner Arber, Daniel
Nathans, and Hamilton
Smith.
4. 4
RESTRICTION SYSTEM
●
REs scan the length of DNA.
●
It identifies specific sequences.
●
Binds to DNA at restriction site.
●
Makes a cut in the sugar-phosphate backbone.
●
Mg2+ acts as a co-factor in this process.
●
Blunt or staggered end cuts are formed.
5. 5
MODIFICATION SYSTEM
●
The sequence specific methylation of host DNA
is called as modification.
●
Restriction functions only on unmethylated host
DNA.
●
This is what protects the host from its own REs.
●
Modification is done by the methyltransferase
domain of the REs.
6. 6
NOMENCLATURE
●
First letter derived from genus.
●
Next two comes from the specific species.
●
Next letter is the name of the strain.
●
The final letter tells you the order of identification
of the enzyme in the bacteria.
●
Eg: EcoRI, HindIII, BamHI etc.
7. 7
Types of Restriction Enzymes
Type of RE Type I Type II Type III Type IV
Features Oligomeric
REase and
MTase complex.
Require ATP
hydrolysis for
restriction.
Cleave variably,
often far from
recognition site
Separate REase
and MTase or
combined
REase MTase∼MTase
fusion.
Cleave within or
at fixed
positions close
to recognition
site.
Combined
REase + MTase
complex.
ATP required for
restriction.
Cleave at fixed
position outside
recognition site.
Methylation-
dependent
Rease.
Cleave at
variable distance
from recognition
site.
Cleave m6A,
m5C, hm5C
and/or other
modified DNA.
Genes hsdR, hsdM,
hsdS
ecorIR, ecorIM ecoP1IM,
ecoP1IR
mcrA, mcrBC,
mrr
Examples EcoK1 EcoR1 EcoP1I No typical
examples
8. 8
FUNCTIONAL ROLES OF R-M SYSTEM
●
,
S MT
ET M
SEQUENCE SPECIFICITY METHYL TRANSFERASE
TRANSLOCATION SAM-BINDING ENDONUCLEASE
9. 9
TYPE I ENDONUCLEASES
●
First to be identified by Arber and Meselson.
●
Asymmetric recognition sequence.
●
Requires various co-factors including SAM, ATP and Mg2+.
●
Single enzyme that performs restriction and modification
functions.
●
Contains 3 subunits HsdS, HsdM and HsdR.
10. 10
TYPE III ENDONUCLEASES
●
Cleave DNA at immediate vicinity, about 20-30 base pairs away
from recognition sequence.
●
Recognises two separate non-pallindromic sequences that are
inversely oriented.
●
ATP, SAM (not essential) and Mg2+ acts as co-factor.
●
Separate enzymes for restriction and modification, but share a
common subunit.
11. 11
TYPE II ENDONUCLEASES
●
First identified in 1970 (HindII).
●
Most commonly used in genetic manipulation experiments.
●
Recognizes 4-8 bp long pallindromic sequences.
●
Cleaves within (mostly) the recognition sequence.
●
Only Mg2+ required as cofactor, doesn’t require SAM or ATP for
function.
12. 12
How Type II Res work?
FIGURE: Mechanism of type II RE action
SOURCE: Pingoud and Jeltsch, 2001
13. 13
Applications of Type II REs
●
Gene cloning and protein expression experiments.
●
Restriction mapping and vector designing.
●
Study fragment length differences among
individuals. Eg: RFLP, AFLP.
14. 14
Previous Years Entrance Questions
Question 1: Which one of the following marker types uses
combination of both restriction enzymes and PCR
techniques? (CSIR-NET: June 2014, Part-B)
●
SSR
●
AFLP
●
SNP
●
RAPD
20. 20
REFERENCES:
●
Horton, J. et al. (2006). Structure and substrate recognition of the Escherichia coli DNA
adenine methyltransferase. Journal of Molecular Biology, 358, 559–570.
●
Kennaway, C. et al. (2012). Structure and operation of the DNA-translocating type I DNA
restriction enzymes. Genes Development, 26(1), 92-104.
●
Leonen, W. et al. (2014). Highlights of the DNA cutters: a short history of the restriction
enzymes. Nucleic acids research, 42(1), 3-19.
●
Pingoud, A. and Jeltsch, A. (2001). Structure and function of type II restriction endonucleases.
Nucleic acids research, 29(18), 3705-3727.
●
Pingoud, A. et al. (2014). Type II restriction endonucleases:a historical perspective and more.
Nucleic acids research, 42(12), 7489-7527.
●
Roberts, R. (2005). How restriction enzymes became the workhorses of molecular biology.
Proceedings of the National Academy of Sciences of the United States of America, 102(17),
5905-5908.
●
Toliusis, P. (2017). CgII cleaves DNA using a mechanism distinct from other ATP-dependent
restriction endonucleases. Nucleic acids research, 45(14), 8435-8447.
