Restriction endonucleases

Restriction Endonucleases are enzymes from bacteria that can recognize specific base sequences in DNA and cut (restrict) the DNA at that site (the restriction site). This powerpoint sllides illustrate the introduction, examples, nomenclature and types of restriction endonucleases.

2. Restriction enzymes areDNA-cutting enzymesfound in
bacteria. Because they cut within the molecule, they are
often calledrestriction endonucleases.
Enzymes that recognize a specific DNA sequence, called
arestriction site, and cleave the DNA within or adjacent
to that site.
Restriction
EndonucleaseEcoRI

3. EcoRI cleaves the DNA between the G and A on each strand,
producing 5′overhangs of four nucleotides, as shown here.
The termini produced byEcoRI, since they are
complementary at their single-stranded overhangs, are said to
becohesiveorsticky.

4. Restriction enzymes were discovered and characterized in the
late 1960s and early 1970s by molecular biologists
Werner Arber,Hamilton O. Smith, andDaniel Nathans.

5. 1. Each RE enzyme is named by a three-letter code.
2. The first letter of this code is derived from the first epithet (first
letter of name) of the genus name. It is printed in italics.
3. The second and third letters are from the first two letters of its
species name. They are also printed in italics.
4. This is followed by the strain number. If a particular strain has
more than one restriction enzyme, these will be identified by
Roman numerals as I, II, III, etc.

6. For example,
the enzyme Eco RI was isolated from the bacterium
Escherichia (E)
coli (co)
strain RY13 (R) and
it was the first endonuclease (I).
R also indicates antibiotic resistant plasmid of the
bacterium.
Likewise, Hind II from Haemophilusinfluenzaestrain Rd
and BglI from Bacillus globigii.

7. specific sequences of nucleotides recognisedby RE.
The recognition squencescan also be classified by the number of
bases in its recognition site, usually between 4 and 8 bases, and
the number of bases in the sequence will determine how often the
site will appear by chance in any given genome,
e.g., a 4-base pair sequence would theoretically occur once every
4^
4
or 256 bp,
6 bases, 4^
6
or 4,096bp, and
8 bases would be 4^
8
or 65,536bp.
Many of them arepalindromic, meaning the base sequence reads
the same backwards and forwards.

8. Twotypesofpalindromicsequences
1.Themirror-likepalindrome-
Itissimilartothosefoundinordinarytext,inwhichasequence
readsthesameforwardandbackwardonasinglestrandofDNA,
asinGTAATG.
2.Theinvertedrepeatpalindrome-
Itisalsoasequencethatreadsthesameforwardandbackward,
buttheforwardandbackwardsequencesarefoundin
complementaryDNAstrands(i.e.,ofdouble-strandedDNA),as
inGTATAC(GTATACbeingcomplementarytoCATATG).
Invertedrepeatpalindromesaremorecommonandhavegreater
biologicalimportancethanmirror-likepalindromes.

9. EcoRIdigestion produces"sticky" ends
SmaIrestriction enzyme cleavage produces"blunt" ends

10. Two types of cut ends of DNA
A) blunt or flush ends and
B) sticky or cohesive ends
Blunt ends
are formed by a straight cut
do not contain any unpaired bases or overhangs at the 3′or 5′regions.
Hence, it is difficult to ligate the fragments with blunt ends.
Sticky ends
are generated by a staggered cut
have unpaired bases or overhangs at the 3′and 5′regions.
These overhangs are useful during the ligation as they ensure proper
joining of the fragments.

11. Nature of Cut Ends

12. Sticky end→the end of
nitrogenousbasesthat
have hydrogen bonds to
pair up with the plasmid
DNA (the DNA to be
inserted via the vector).
Blunt end→the end
with lesser number of
nitrogen base that gets
ligasedto the new
plasmid DNA.

13. Restriction enzymes are traditionally classified into
four types on the basis of subunit composition,
cleavage position, sequence specificity and
cofactor requirements.
There are four classes of restriction endonucleases:
types I, II,III and IV.

14. Type I enzymes are complex, multi subunit, combination
restriction-and-modification enzymes that cut DNA at
random far from their recognition sequences i.ecleave at
sites remote from a recognition site;
It requires both ATP and S-adenosyl-L-methionine to
function; multifunctional protein with both restriction and
methylaseactivities.
They have little practical value since they do not produce
discrete restriction fragments or distinct gel-banding
patterns.

15. Type II enzymes cut DNA at defined positions close to or
within their recognition sequences.
They produce discrete restriction fragments and distinct gel
banding patterns, and they are the only class used in the
laboratory for routine DNA analysis and gene cloning.
It most requires magnesium; single function (restriction)
enzymes independent of methylase.

16. Type III enzymes cleave at sites a short distance from a
recognition site;
require ATP (but do not hydrolyseit);
S-adenosyl-L-methionine stimulates the reaction but is not
required;
exist as part of a complex with a modification methylase.

17. Type IV enzymes target modified DNA,
e.g. methylated, hydroxymethylatedand glucosyl-
hydroxymethylatedDNA

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