ENZYMES AS TOOLS IN GENE MANIPULATION

ENZYMES AS TOOLS IN GENE MANIPULATION
Enzymes that break DNA and RNA backbones:
1. Endonucleases – degrade nucleic acids from internal sites: restriction enzymes,
deoxyribonucleases, ribonucleases
2. Exonucleases – degrade nucleic acids from 5´ end or the 3´ end
3. Endo-exonucleases – degrade DNA and/or RNA both from internal and terminal
positions
Enzymes that join DNA and RNA molecules – ligases
Enzymes that synthesize new DNA and RNA backbone bonds:
1. DNA polymerases: synthesize DNA strand in the 5´ to 3´ direction using DNA
strand as a template
2. RNA polymerases: synthesize RNA strand in the 5´ to 3´ direction using DNA
strand as a template
3. Reverse transcriptase: synthesize DNA strand in the 5´ to 3´ direction using RNA
strand as a template
Enzymes that add or remove phosphatase at nucleic acids termini:
1. Kinase: adds phosphatase groups to the 5´ ends of nucleic acid moleculs
2. Phosphatase: removes phosphate groups from nucleic acid termini
RESTRICTION ENZYMES
In 1960s host-controlled restriction and modification system was characterized:
The restricted growth of bacterophage λ was observed when phage was transferred
between different E.Coli strains – due to the presence of degradative nucleases in the
host cell that destroyed the ivading phage DNA.
Modification: in order to prevent destruction of its own DNA by the restriction
enzymes, the bacterium marks its own DNA by methylation.
The first restriction endonuclease isolated: 1968 from E.Coli strain K (Meselson and
Yuan) and strain B (Linn and Arber).
TYPE II RESTRICTION ENDONUCLEASES
Recognize a specific target sequence in a double-stranded DNA and break the DNA strand
within or near to that sequence giving rise to DNA fragments of defined length, all having
identical termini.
The first type II restriction enzyme was isolated in 1970 from Haemophilus influenzae Rd
strain.
Nomenclature: the naming derives from the microorganism that was a source of the activity:
EcoRI – E.Coli strain R, chronologically the first (I) endonuclease recovered from this source
EcoRII, EcoRIII, EcoRIV and EcoRV – later isolated restriction endonucleases with distinct
activities from the same E.Coli strain.
HindI, HindII, HindIII, etc. – H.influenzae strain Rd restriction endonuclease activities.
More than 1000 restriction enzymes representing more than 120 different specificities have
been isolated.
TARGET SITES OF TYPE II RESTRICTION ENZYMES
The vast majority of type II enzymes recognize and break a double stranded DNA sequence
that possesses a twofold axis of rotational symmetry, reading the same 5´ to 3´ direction
along the upper strand as it does along the lower strand:
Such sequences are termed palindromes.
PROPERTIES OF EcoRI CLEAVAGE
The enzyme makes single-stranded breaks 4 nucleotide pairs apart in the opposite strands of
its target sequence:
The generated overlapping ends are referred as sticky or cohesive ends because their ability
to re-form a duplex, base-paired structure with another DNA fragment bearing a
complementary sequence at its ends.
EcoRI cleaves DNA so that a free 3´-hydroxyl group and a 5´-phosphoryl group are formed:
Only one restriction enzyme, NciI cleaves so that the DNA fragments produced possess a
phosphate group at the 3´ end and a hydroxyl group at 5´ end.
Recognition Sequences, Substrate cleavage frequencies, buffer, assay, and heat
inactivation properties of the restriction enzymes
(a) The generic buffer all contain 40 mM TrisHCl, Ph 7.5/10Mm dithiotreitol. Those designated as L, M and
H contain 0, 50, and 100Mm NaCl, respectively. Those designated with a K contain KCl rather than
NaCl.
(b) Heat inactivation measures the amount of enzyme activity remaining after 15 minute incubation at
65°C. Yes, partial, and no reflect >90%, >50%, and <10% inactivation, respectively.
(c) Numbers in parantheses indicate cleavage downstream from the cognate sequence. The first number
refers to the upper strand and the second number to the position on the lower strand.
THREE DIFFERENT WAYS OF CLEAVAGE
CATALYZED BY THE TYPE II RESTRICTION ENZYMES
ISOSCHIZOMERS
Restriction enzymes isolated from different microorganisms that recognize the same
sequence and may or may not cleave that sequence in different ways.
In many cases isoschizomers cleave DNA at the same sequences in the same way:
In some cases the DNA fragments generated will have non-identical termini because
isischizomers cleave in target sequence in different ways:
ISOCAUDOMERS
Group of restriction enzymes isolated from different microorganisms that recognize
different sequences, but cleave these sequences in such a way that the created termini are
identical.
COMPATIBLE ENDS CAN BE RELIGATED
TYPE II RESTRICTION ENZYMES THAT DO NOT
PRODUCE FRAGMENTS WITH IDENTICAL ENDS
Example: BglI
The enzyme produces fragments with heterogenous ends, leaving 3 NNN residues
protruding at the 3´ terminus.
METHYLATION
Many E.Coli strains posess dam and dcm methylation activities:
Dam: DNA adenosine methylase, creates 6-methyladenine in the GATC sequence
Dcm: DNA cytosine methylase, creates 5-methylcytosine in the CC(A/T)GG sequence
How to overcome problems associated with DNA methylation?
Try to find isoschizomer that is insensitive to methylation:
Example: dam methylation of the sequence of GATC renders it resistant to restriction by
MboI; isoschizomer Sau3AI cleaves this methylated sequence.
Use dam- or dcm- strain of E.Coli
DEOXYRIBONUCLEASE I (DNase I)
Endonuclease, isolated from bovine pancreas: degrades DNA by hydrolyzing internal
phosphoester linkages to produce predominantly dinucleotides of general formula
p(Py)p(Pu), oligonucleotides with 5´-phosphate and 3´-hydroxyl groups, and less often
nucleoside 5´-monophosphates.
Prefers native, duplex DNA
Applications:
 to probe the structure of chromatin – regions of chromatin that are actively
transcribed are more open and less constrained by histone interactions and are
therfore more susceptible to DNase digestion; DNase I hypersensitivity has been
accepted as a measure of gene activity within a DNA region.
 To clone random DNA fragments into vectors possessing blunt ends.
 DNA „footprinting“ technique: allows to analyze the specific stretch of DNA
residues that are associated with a protein factor: DNA associated with proteins is
protected from DNase I digestion.
RIBONUCLEASES (RNase A, T1, H)
RNase A: isolated from bovine pancreas, also named bovine ribonuclease. Exhibits two
disparate activities:
 A ribonuclease that cleaves 3´ to pyrimidine residues to yield oligonucleotides
terminating in C or U
 a DNA unwinding protein; derived from its affinity to bind to single-stranded DNA
Applications:
 a common general ribonuclease for destroying RNA that may be contaminating a
DNA sample
 a pyrimidine-specific nuclease for RNA sequence analysis
RNase T1: a guanosine-specific ribonuclease that cleaves 3´ side of G residues to yield
fragments terminating with Gp.
Applications: a G-specific endoribonuclease for RNA sequence analysis
RNase H: degrades specifically the RNA strand in DNA:RNA heteroduplexes, producing 5´phosphorylated mono- and oligonucleotides; does not cleave RNA:RNA or DNA:DNA
duplexes.
Applications:
 to introduce site-specific cleavage of RNA to yield oligoribonucleotide fragments that
can be end-labeled and used subsequently for RNA sequence analysis
 to generate short oligoribonucleotide primers for second-strand synthesis of cDNA
NUCLEASE S1
Endo- and exonuclease: degrades RNA or single-stranded DNA into nucleoside 5´monophosphates, being approximately 5 times more effective degrading single-stranded
DNA than RNA.
Does not degrade duplex DNA or RNA:DNA heteroduplex.
Applications:
 to define the 5´ end or transcriptional start site for the mRNA when genomic
sequence is known
 to map exon-intron boundaries
 to digest hairpins in double-stranded cDNA during cDNA synthesis
LIGASES
ENZYMES THAT SYNTHESIZE
DNA AND RNA BACKBONE BONDS
DNA POLYMERASE I
LARGE FRAGMENT POLYMERASE I –
KLENOW POLYMERASE
T7 DNA POLYMERASE
Taq DNA POLYMERASE
RNA POLYMERASE
REVERSE TRANSCRIPTASE
TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE
ENZYMES THAT ADD PHOSPHATE GROUPS TO THE 5´
ENDS OF NUCLEIC ACID MOLECULES: KINASES
ENZYMES THAT REMOVE PHOSPHATE GROUPS TO THE 5´
ENDS OF NUCLEIC ACID MOLECULES: PHOSPHATASES
SUMMARY