Restriction endonucleases

 Restriction enzymes are DNA-cutting enzymes found in
bacteria. Because they cut within the molecule, they are
often called restriction endonucleases.
 Enzymes that recognize a specific DNA sequence, called
a restriction site, and cleave the DNA within or adjacent
to that site.
Restriction
Endonuclease EcoR I

 EcoR I cleaves the DNA between the G and A on each strand,
producing 5′ overhangs of four nucleotides, as shown here.
 The termini produced by EcoR I, since they are
complementary at their single-stranded overhangs, are said to
be cohesive or sticky.

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

 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.

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 Haemophilus influenzae strain Rd
and Bgl I from Bacillus globigii.

 specific sequences of nucleotides recognised by RE.
 The recognition squences can 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 are palindromic, meaning the base sequence reads
the same backwards and forwards.

 Two types of palindromic sequences
 1. The mirror-like palindrome-
 It is similar to those found in ordinary text, in which a sequence
reads the same forward and backward on a single strand of DNA,
as in GTAATG.
 2. The inverted repeat palindrome -
 It is also a sequence that reads the same forward and backward,
but the forward and backward sequences are found in
complementary DNA strands (i.e., of double-stranded DNA), as
in GTATAC (GTATAC being complementary to CATATG).
 Inverted repeat palindromes are more common and have greater
biological importance than mirror-like palindromes.

EcoRI digestion produces "sticky" ends
SmaI restriction enzyme cleavage produces "blunt" ends

 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.

Sticky end→ the end of
nitrogenous bases that
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
ligased to the new
plasmid DNA.

 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.

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

 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.

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

 Type IV enzymes target modified DNA,
 e.g. methylated, hydroxymethylated and glucosyl-
hydroxymethylated DNA

Restriction endonucleases

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Restriction endonucleases