Restriction Enzymes
Gabriela Perales
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Restriction Enzymes
Restriction enzymes, also called restriction endonucleases,
are molecules that cut double stranded DNA at specific
sites
Cut by hydrolysis (digestion) of the phosphodiester
backbone
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Restriction Enzymes
Were discovered about 40 years ago during investigations
of the phenomenon of host-specfic restriction and
modification of bacterial viruses.
Bacteria resist infections by new viruses and this
restriction of viral growth stems from endonucleases
within the cells that destroy foreign DNA molecules.
Restriction enzymes protect bacteria from infections by
viruses. They function as microbial immune systems.
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Restriction Enzymes
Some of the first restriction enzymes to be purified were
EcoRI and EcoRII from Escherichia coli and Hind II and
HindIII from Haemophilus influenzae.
In 1978, the Nobel Prize was awarded jointly to Werner
Arber, Daniel Nathans, and Hamilton O. Smith for the
discovery of restriction enzymes and their application to
molecular genetics.
In 1980’s, restriction enzymes began to be isolated and
purified commercially by the New England BioLabs
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Naming Restriction Enzymes
The enzyme name is comprised of the first letter of the
genus name and the first two letters of the species name.
Escherichia coli = Eco
The strain or identification follows.
RY13=EcoR
when a strain has more then one restriction enzyme, they
are numbered in the order they were discovered.
EcoR 1
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Restriction Enzymes
Restriction enzymes are found only in bacteria and archaea
They range in size from Pvu II(157 amino acids) to Cje I (1250
amino acids).
Restriction enzymes usually occur in combination with other
modification enzymes that protect the cell’s own DNA from
cleavage by the restriction enzyme.
These modification enzymes recognize the same DNA as the
restriction enzyme they accompany, but instead of cleaving the
sequence, they methylate one of the bases in each of the DNA
strands.
The methyl group extends into the major grove of DNA at the
binding site and prevent the enzyme from acting upon it.
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Restriction Enzymes
Restriction enzymes are traditionally classified into three
categories, (Type I, II, and III) on the basis of subunit
composition, cleavage position…
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Type I Enzymes
Complex multi-subunit that cut DNA at random far from
their recognition sequences.
They do not produce discrete restriction fragments or
distinct gel banding patterns so they are not a good
biotech interest.
found in E. coli and its relatives, are the most complex
known. They are comprise of three polypeptides, R
(restriction), M (modification), and S (specificity).
The resulting complex is both an endonuclease and a
methyltransferase that work together.
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Type II Enzymes
Cut DNA at defined positions that are close or within
the recognition sequences.
Produce discrete restriction fragments and distinct gel
patterns.
Are composed of a collection of different proteins that
greatly differ in their amino acid sequence.
These are that ones that are used in a laboratory for
DNA analysis and gene cloning.
Endonucleases and methyltransferases act independently
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Type II Enzymes
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Most common type of enzymes cleave within the
recognition sequences and are symmetric
5’…AˇAGCTT…3’
3’…TTCGAˆA…5’
Other types cleave outside recognition sequence and are
symmetric and interrupted.
5’…CCA(N)9ˇTGG…3’
3’…GGT(N)9ˆACC…5’
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Type III Enzymes
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Composed of large combination of R-M enzymes.
They cleave outside their recognition sequences and need
two sequences in opposite orientation in order to cleave.
Not used in laboratories.
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Recognition Sites
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Restriction enzymes bind to DNA at a specific sequence
This sequence is usually 4 to 8 bases pairs long.
These sequences form an inverted repeat called a
palindrome
5’…GˇAATTC…3’
3’…CTTAAˆG…5’
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Sticky Ends vs Blunt Ends
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Depending on where and how the enzyme cuts it can
leave a blunt end or sticky end.
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Applications
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Recombining DNA
Methylation-Specific Restriction Digestion
Restriction Fragment Length Polymorphism Analysis
(RFLP)
Fingerprinting
Cloning
In class: cloned copies of bacteria that contained our PCR
product in their plasmids; restriction enzymes were most
likely used to cleave the plasmid and insert PCR product
in the recognition sites of a specific restriction enzyme.
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