Unit 1 (continuation):

Restriction Enzymes for

Gene Manipulation and Genomics

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13/07/2018
 RESTRICTION ENDONUCLEASES
AND DNA MODIFYING ENZYMES
Restriction endonucleases are enzymes that cleave the
sugar-phosphate backbone of DNA.
In most practical settings, a given enzyme cuts both
strands of duplex DNA within a stretch of just few
bases.
Several thousand different restriction endonucleases
have been isolated, which collectively exhibit a few
hundred different sequence (substrate) specificities.
Type II Endonucleases find application in molecular
biology. 2
 Cuts Produced by Restriction Endonucleases

Type IV enzymes recognize modified, typically methylated DNA.

Type V restriction enzymes utilize guide RNAs to target specific non-palindromic sequences.
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Restriction-Modification Systems
A large majority of restriction enzymes have been
isolated from bacteria, where they appear to serve a
host-defense role.
The idea is that foreign DNA, for example from an
infecting virus, will be chopped up and inactivated
("restricted") within the bacterium by the restriction
enzyme.
In almost all cases, a bacterium that makes a particular
restriction endonuclease also synthesizes a companion
DNA methyltransferase, which methylates the DNA
target sequence for that restriction enzyme, thereby
protecting it from cleavage.
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 This combination of restriction endonuclease and
methylase is referred to as a restriction-modification
system.

By convention, restriction enzymes are named after

their host of origin. For example, EcoRI was isolated
from Escherichia coli (strain RY13), Hind II and Hind
III from Haemophilus influenzae, and XhoI from
Xanthomonas holcicola.

(Escherichia coli ("Eco"); strain RY13 ("R"), restriction

endonuclease number "I“)
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 Properties of Restriction Enzymes:
1. Recognition sequences
The substrates for restriction enzymes are specific
sequences of double-stranded DNA.

Most recognition sites for

commonly used restriction
enzymes are palindromes.
(nitrogenous base sequences that read the
same backwards and forwards)

Most restriction enzymes bind

to their recognition site as dimers
(pairs).
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The length of restriction recognition sites varies. For
example, the enzymes EcoRI, SacI and SstI each
recognizes a 6 base-pair (bp) sequence of DNA,
whereas NotI recognizes a sequence 8 bp in length,
and the recognition site for Sau3AI is only 4 bp in
length.

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List of rest. endonucleases & their cleavage sites
Enzyme Site Enzyme Site
AluI AG'CT NotI GC'GGCCGC
BamHI G'GATCC PstI CTGCA'G
BglII A'GATCT PvuII CAG'CTG
EcoRI G'AATTC SalI G'TCGAC
HaeIII GG'CC Sau3AI 'GATC
HhaI GCG'C SmaI CCC'GGG
HincII GTY'RAC SpeI A'CTAGT
HindIII A'AGCTT TaqI T'CGA
HinfI G'ANTC XbaI T'CTAGA
HpaII C'CGG XhoI C'TCGAG
KpnI GGTAC'C XmaI C'CCGGG
MboI 'GATC 8
2. Isoschizomers

Different restriction enzymes with the same

recognition site.

Recognition sites for SacI and SstI are identical.

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3. Restriction recognition sites can be ambiguous
or unambiguous

The enzyme BamHI recognizes the sequence GGATCC

and no others - unambiguous.

In contrast, HinfI recognizes a 5 bp sequence starting

with GA, ending in TC, and having any base between
(in the table, "N" stands for any nucleotide) –
ambiguous (having more than one).

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Patterns of DNA Cutting by Restriction Enzymes:
The restriction enzymes most used in molecular biology
labs cut within their recognition sites and generate one
of three different types of ends.
5' overhangs: The enzyme cuts asymmetrically within the
recognition site such that a short single-stranded segment
extends from the 5' ends. BamHI cuts in this manner.

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3' overhangs: The enzyme cuts asymmetrically within the
recognition site, resulting in a single-stranded overhang
from the two 3' ends. KpnI cuts in this manner.

Blunts: Enzymes that cut at precisely opposite sites in the

two strands of DNA generate blunt ends without
overhangs. SmaI is an example that cuts in this manner.

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Addition of Restriction enzyme site in the DNA
Linkers are short duplex oligonucleotides that
contain a restriction endonuclease cleavage site. They
can be ligated onto any blunt‑ended molecule,
thereby generating a new restriction cleavage site on
the ends of the molecule.

Ligation of a linker on a restriction fragment

followed by cleavage with the restriction
endonuclease is one of the several ways to generate an
end that is easy to ligate to another DNA fragment.

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 Ligation and Digestion of Linker:
Introducing A Restriction Enzyme Site

NNNN Blunt end - before

NNNN addition of linker
GCCGGAATTCCGGNNNNN
After ligation of linker
CGGCCTTAAGGCCNNNNN
AATTCCGGNNNNN
After digestion of linker
GGCCNNNNN

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 Homopolymer tailing

Annealing of homopolymer tails is another way to join two

different DNA molecules.

The enzyme terminal deoxynucleotidyl transferase will catalyze

the addition of a string of nucleotides to the 3' end of a DNA
fragment.

Terminal transferase (TdT) is a template independent

polymerase that catalyzes the addition of deoxynucleotides to
the 3' hydroxyl terminus of DNA molecules.

Protruding, recessed or blunt-ended double or single-stranded

DNA molecules serve as a substrate for TdT. The 58.3 kDa
enzyme does not have 5' or 3' exonuclease activity.
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 By incubating each DNA fragment with the appropriate dNTP
and terminal deoxynucleotidyl transferase, one can add
complementary homopolymers (GC or AT) to the ends of the
DNAs that one wants to combine.

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