Enzimas I

Enzimas de Restricción
• Restriction enzymes, also known as restriction endonucleases, are enzymes that
cut a DNA molecule at a particular place. They are essential tools for recombinant
DNA technology. The enzyme "scans" a DNA molecule, looking for a particular
sequence, usually of four to six nucleotides. Once it finds this recognition
sequence, it stops and cuts the strands. This is known as enzyme digestion. On
double stranded DNA the recognition sequence is on both strands, but runs in
opposite directions. This allows the enzyme to cut both strands. Sometimes the
cut is blunt, sometimes the cut is uneven with dangling nucleotides on one of the
two strands. This uneven cut is known as sticky ends.

Extremo Romo

Extremo Cohesivo
 RESTRICTION MODIFICATION SYSTEMS OF BACTERIA

Restriction/modification systems were first described based

upon observations of host-specificity in bacteriophage
infections.
Certain phage appeared to be RESTRICTED to certain
host strains (the ones from which they came).

PHAGE INFECT INFECT

FROM STRAIN A STRAIN B

Strain A LYSIS NO LYSIS

Strain B NO LYSIS LYSIS

It appeared as if certain strains were "immune" to infection by certain

bacteriophage, while other strains were "immune" to infection by different
bacteriophage.
REASON:
Many bacterial strains carry restriction-modification
systems that recognize foreign DNA (as in phage DNA) and cleave
it to prevent infection.
At the same time, these systems protect the host cell
chromosomal DNA from similar cleavage and degradation.
Restriction enzymes recognize and cleave DNA at specific
sequences (like palindromes).
The distinction between "foreign" and "self" DNA is made
through a specific modification (METHYLATION) of the bacterial
DNA, which protects it from cleavage by the restriction enzyme
carried by the bacterium.
The same sequence is a target for both restriction
(cleavage) by the endonuclease, and methylation by the methylase.
Methylated restriction sites are protected from cleavage.
Restriction Enzymes
WHY IS THERE HOST-SPECIFICITY?
Different bacterial strains, such as A and B,
have different recognition sequences for restriction
and modification.
Therefore, while strain A DNA may be
methylated and protected at a specific sequence,
strain B host cells would produce a restriction
enzyme that recognizes and cleaves a DIFFERENT
SEQUENCE.
Thus, when phage from strain A infect strain B,
the strain B restriction enzyme degrades the invading
DNA.
Why isn’t invading phage DNA methylated upon entry into
the host, just as host DNA is?
ONLY HEMIMETHYLATED DNA is a target for
methylation
Following replication, host DNA is hemimethylated
This hemimethylated DNA is a target for the
methylase, which results in the production of fully
methylated DNA
Fully methylated DNA is protected from restriction
Invading phage DNA would be fully unmethylated,
which is a target for restriction by the endonuclease
MODIFIED BAS ES
Protection of DNA cleavage by modification. The restriction enzyme EcoRI recognizes and cleaves the
sequence 5-GAATTC-3. E. coli R strains, from which this enzyme is derived, protect their own DNA from
fragmentation by also producing a specific methylase. EcoRI methylase takes a methyl group from S-
adenosylmethionine and places it on the N6 of the second adenine in the recognition sequence. EcoRI
restriction enzyme is unable to cleave the methylated DNA.
 Restriction Enzymes
• Also known as restriction endonucleases
• Scan the DNA sequence
• Find a very specific set of nucleotides
• Make a specific cut
• Used to construct recombinant DNA plasmids
 Palindromes in DNA sequences
Genetic palindromes
5 3’ are similar to verbal
’
palindromes. A
palindromic sequence
in DNA is one in which
the 5’ to 3’ base pair
3’ 5
’ sequence is identical
on both strands.
Restriction enzymes recognize and
make a cut within specific
palindromic sequences, known as
restriction sites, in the DNA. This is
usually a 4- or 6 base pair sequence.
 Restriction Endonuclease Types
Type I- multi-subunit, both endonuclease and
methylase activities, cleave at random up to
1000 bp from recognition sequence
Type II- most single subunit, cleave DNA within
recognition sequence
Type III- multi-subunit, endonuclease and
methylase about 25 bp from recognition
sequence
TYPE III RM systems are interesting in that the M and S
function is found in one protein labeled MS (hsdMS ---> 73-
80kd in E.coli) while the restriction is accomplished by the
hsdR gene product R (hsdR ---> 106-110kd in Ec). The
recognition site is a 5-7 bp asymmetrical sequence. Cleavage
is ATP dependent 24-26 base pairs downstream from the
recognition site and usually yields staggered cuts 2-4 bases
apart.
TYPE II RM systems are the simplest and most common
(at least commercially). In this system, there are separate
R and M proteins each able to recognize the cleavage site
(no S protein needed). Each may compete directly and no
complex is formed. Site recognition is via short
palindromic base sequences that are 4-6 base pairs long.
Cleavage is at the recognition site (but may occasionally
be just adjacent to the palindromic sequence, usually
within) and may produce blunt end terminuses or
staggered, "sticky end" terminuses. No ATP requirements
have been detected for the endonuclease activity.
 Hae III
HaeIII is a restriction enzyme that
searches the DNA molecule until it finds
this sequence of four nitrogen bases.

5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
Once the recognition site is found Hae III will
cleave the DNA at that site

5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
 These cuts produce
“blunt ends”

5’ TGACGGGTTCGAGG CCAG 3’
3’ ACTGCCCAAGGTCC GGTC 5’
 The names for restriction enzymes come from:

• the type of bacteria in which the enzyme is found

• the order in which the restriction enzyme was identified
and isolated.

EcoRI for example

R strain of E.coli bacteria
I as it is was the first E. coli restriction enzyme to
be discovered.
Each restriction enzyme is named based upon the specific strain from which it was isolated. For example:

Enzyme Isolated From: Recognition

Seq.

EcoRI E. coli, strain R, 1st G AATTC

enzyme

EcoRV E. coli, strain R, 5th GATATC

enzyme

HindIII H. influenzae, strain A AGCTT

d, 3rd enzyme
“blunt ends” and “sticky ends”
Hae III produced a “blunt end”?
EcoRI makes a staggered cut and produces a
“sticky end”
5’ GAATTC 3’
3’ CTTAAG 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G AATTC 3’
3’ CTTAA G 5’
More examples of restriction sites of restriction
enzymes with their cut sites
Hind III: 5’ AAGCTT 3’
3’ TTCGAA 5’

Bam HI: 5’ GGATCC 3’

3’ CCTAGG 5’

Alu I: 5’ AGCT 3’
3’ TCGA 5’
 How Do Type II Restriction Enzymes Work?

The structure of the restriction enzyme BamHI bound to DNA. The enzyme recognizes double-
stranded DNA of the sequence 5-GGATCC-3, and cleaves the phosphodiester backbone between
the two G residues. This results in formation of two DNA fragments that have 5 overhanging
ends – called cohesive or sticky ends. The protein is a dimer of identical subunits (coloured in
green and cyan)
DNA cleavage by type II restriction enzymes. Different restriction enzymes recognize
different DNA sequences and can cleave to give either overhanging ends or blunt ends. The
recognition site of each enzyme is shown, together with the cleavage site, indicated by the
blue line. After cleavage, the weak hydrogen bonds holding the overhanging ends will fall
apart, and two new DNA ends will be formed
Separating Restriction Fragments
Our digests:
…
Run digest on gel to check fragment sizes
• miR-29c construct ~ 5kB plasmid
– BglII/EcoRI digest ~250bp fragment

• PRKCBP1 construct ~9kB plasmid

– AsiSI/BglII digest ~1.8kB fragment
 Restriction maps
• After digest with DNA restriction endonuclease the fragments can be
separated according to size by gel electrophoresis.
• DNA can be separated according to size by agarose or polyacrylamide.
• Duplex DNA is detected by staining with intercalating, planar, aromatic
cations such as ethidium, acridine orange, or proflavin, between
stacked base pairs. New stains like SYBR are available that are
notThese exhibit flurorescence under UV light.
• As little as 50 ng of DNA may be detected in a gel by staining it with
ethidum bromide.
• Can also be used to visualize single stranded DNA or RNA.
• Can be used to generate a restriction map.
 Figure 5-38 Agarose gel electrophoretogram of restriction
digests.
Digest of Agrobacterium radiobacter plasmid
pAgK84 digested with:
23130 bp
A. BamHI, B. PstI, C. BglII, D. HaeIII, E. HincII, F.
SacI, G. XbaI, H. HpaI. Lane I contains l phage
9416 bp
DNA digested with HindIII as standards.
6557 bp

4361 bp

2322 bp
2027 bp
Page 103
 Figure 5-39 Construction of a restriction map.
Page 104
 Figure 5-40 Restriction map for the 5243-bp circular DNA
of SV40.
Page 104
 Restriction-fragment length polymorphisms

• Individuality in species derives from genetic

polymorphism; homologous human chromosomes differ in
sequence ~every 1250 bp.
• Restriction enzyme digests of the corresponding segments
from homologous chromosomes contain fragments of
different lengths (restriction fragment length
polymorphisms or RFLPs) which can be used for
identification.
 Figure 5-41 Restriction-fragment length polymorphisms.
Page 105
 Uses of restriction enzyme analysis- forensics

http://science.howstuff
works.com/dna-
profiling1.htm
Uses of restriction enzyme analysis-
Medical diagnosis

http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/
Uses of restriction enzyme analysis-
Inheritance and genetic counseling

http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/
Isoschizomers
Star Activity
When DNA is digested with certain restriction enzymes under
non-standard conditions, cleavage can occur at sites different
from the normal recognition sequence - such aberrent cutting is
called "star activity". An example of an enzyme that can exhibit
star activity is EcoRI; in this case, cleavage can occur within a
number of sequences that differ from the canonical GAATTC by
a single base substitutions.
So what constitutes non-standard conditions? Examples that may
induce star activity include:
•High pH (>8.0) or low ionic strength (e.g. if you forget to add
the buffer)
•Glycerol concentrations > 5% (enzymes are usually sold as
concentrates in 50% glycerol)
•Extremely high concentration of enzyme (>100 U/ug of DNA)
•Presence of organic solvents in the reaction (e.g. ethanol,
DMSO)