Chapter 3: Important enzymes in genetic engineering and molecular biology - DNA modifying enzymes - Bacterial Restriction / Modification system ...
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Chapter 3: Important enzymes in genetic engineering and molecular biology.
- DNA modifying enzymes
- Bacterial Restriction / Modification system
Genetic Engineering 3 Enzymes
Restrictionendonucleases as protection system Genetic Engineering 3 Enzymes
Single Stranded Nicks in DNA
Hydrolysis of
this ester bond
OH
O-
O P OH
O
H2O
The restriction/modification system in bacteria is a small-scale immune system
for protection from infection by foreign DNA.
W. Arber and S. Linn (1969)
Plating efficiencies of bacteriophage lambda (λ phage) grown on E. coli strains C,
K-12 and B, when plated on these bacteria:
E. coli strain on which E. coli strain
parental phage had been for plating
grown phage
C K-12 B
*C 1
Restriction/Methylation Enzyme Genetic Engineering 3 Enzymes
Sites of methylation ¾ Typical sites of methylation include the N6 position of adenine, the N4 position of cytosine, or the C5 position of cytosine. ¾ In addition, only a fractional percentage of bases were methylated (i.e. not every adenine was methylated, for example) and these occurred at very specific sites in the DNA.
Sites of methylation cont.
¾ A characteristic feature of the sites of methylation was that they
involved palindromic DNA sequences.
(EcoR1 methylase specificity. Rubin and Modrich, 1977)
Genetic Engineering 3 Enzymes
Sites of methylation / restriction ¾ In addition to possessing a particular methylase, individual bacterial strains also contained accompanying specific endonuclease activities. ¾ The endonucleases cleaved at or near the methylation recognition site. ¾ These specific nucleases, however, would not cleave at these specific palindromic sequences if the DNA was methylated. Genetic Engineering 3 Enzymes
Restriction endonucleases / Restriction-modification system
The combination of a specific methylase and endonuclease functioned as a type of
immune system for individual bacterial strains, protecting them from infection by
foreign DNA (e.g. viruses).
• In the bacterial strain EcoR1, the sequence GAATTC will be methylated at the
internal adenine base (by the EcoR1 methylase).
• The EcoR1 endonuclease within the same bacteria will not cleave the
methylated DNA.
• Foreign viral DNA, which is not methylated at the sequence "GAATTC" will
therefore be recognized as "foreign" DNA and will be cleaved by the EcoR1
endonuclease.
• Cleavage of the viral DNA renders it non-functional.
Genetic Engineering 3 Enzymes
Replication and Methylation:
• replicating host DNA will initially have one strand (parental) methylated
and the other (nascent strand) non-methylated.
• This is recognized as "self" and is not cleaved by the restriction
endonuclease.
• It is subsequently methylated by the host methylase.
¾ Structural and biochemical studies have indicated that for the common R/M
systems (so called type II), the methylase recognizes and methylates one strand
of the DNA duplex, whereas the restriction endonuclease recognizes both
strands of the DNA (i.e. both strands must be non-methylated for recognition). It
is able to do this because it is a homo-dimer protein.
Genetic Engineering 3 EnzymesRestriction endonucleases
Since different bacterial strains and species have potentially different R/M
systems, their characterization has made available over 200 endonucleases with
different sequence specific cleavage sites.
¾ They are one of the primary tools in modern molecular biology for the
manipulation and identification of DNA sequences.
¾ Restriction endonucleases are commonly named after the bacterium from
which it was isolated.
e.g. EcoRI was purified from an Escherichia coli strain
e.g. BamHI was purified from Bacillus amyloliqueraciens
¾ If several different enzymes are purified from one strain, they will have different
numerals after the strain name.
e.g. PvuI and PvuII are different restriction enzymes from the same strain
¾ Restriction enzymes cut DNA into specific fragments
¾ Restriction enzymes recognize specific base sequences in double-stranded
DNA and cleave both strands of the duplex at specific places.Examples of different restriction enzymes
Examples of different restriction enzymes cont.
Examples of different restriction enzymes cont.
Typical “rare cutter“ enzyme
Genetic Engineering 3 EnzymesFrequency of cutting
¾ The utility of restriction endonucleases lies in their specificity and the frequency
with which their recognition sites occur within any given DNA sample.
¾ If there is a 25% probability for a specific base (A; T; G; C) at any given site,
then the frequency with which different restriction endonuclease sites will
occur can be easily calculated (4n or 0,25n, respectively):
Nucleotide Frequency of
Example
Specificity Occurrence
Four (n=4) Alu I 256 (≈ 0.26 Kb)
Five (n=5) Nci I 1024 (≈ 1.0 Kb)
Six (n=6) EcoR I 4096 (≈ 4.1 Kb)
Seven (n=7) EcoO109I 16384 (≈ 16.4 Kb)
Eight (n=8) Not I 65536 (≈ 65.5 Kb)
Thus, on average, any given DNA will contain an Alu I site every 0.25 kilobases, whereas a Not I site occurs
once about every 65.5 kilobases.4-base cutter:
cuts DNA into 256 bp average-sized fragments in a random sequence
every 256 bp: NO
256 bp average-size fragments: YES
Bar = 256 bp
Genetic Engineering 3 Enzymes• Characteristics of restriction enzymes:
1. Cut DNA sequence-specifically
2. Bacterial enzymes; hundreds are purified and available commercially
3. Restriction-modification system Bacteria have enzymes that will cleave foreign
DNA; hence, “restrict” the entry of viral DNA. To prevent the bacteria’s own DNA
from being cut, there is a second enzyme that methylates the same sites
recognized by the restriction enzyme (modifies that site).
4. Named (e.g., EcoRI) for bacterial genus, species, strain, and type
5. Recognize specific 4-8 bp sequences
• sequences have symmetry (they are palindromes)
• after cutting the DNA, the cut ends are either
• blunt
• staggered (overhangs) - cohesive ends facilitate cloning the DNA
6. Frequency of cutting (4n; n = length of restriction site)
• 4-base cutter 44 = 256 bp
• 5-base cutter 45 = 1,024 bp
• 6-base cutter 46 = 4,096 bp
• 8-base cutter 48 = 65,536 bp
Genetic Engineering 3 EnzymesAcitivities of Restriction endonucleases ¾ One unit of activity is typically defined as the amount of enzyme required to digest (or "restrict") one microgram of reference DNA in one hour at 37 °C. ¾ The endonuclease hydrolysis is a spontaneous reaction and does not require addition of ATP. ¾ Reaction buffers for restriction endonucleases usually contain a buffer component (typically 10 mM TRIS buffer around pH 8.0), magnesium salt (often 10 mM MgCl2), a reducing agent (usually 1mM dithiothreitol, or DTT), a protective carrier protein (typically 100 ug/ml bovine serum albumin, or BSA), and salt (sodium chloride). ¾ The biggest determinant of enzyme activity is typically the ionic concentration (NaCl content) of the buffer (low (20 mM), medium (100 mM) or high (250 mM) salt (NaCl) concentrations) ¾ Enzyme digests are typically performed for 1-2 hours at 37 °C. However, quantitative digestion can sometimes only be achieved after extended incubation (i.e. overnight). Genetic Engineering 3 Enzymes
Types of restriction endonucleases
(A) Typ-1-Restriktionsendonucleasen:
benötigen als Co-Substrate ATP, SAM (S-Adenosyl-Methionin)
und Mg2+. Die Hydrolyse erfolgt weit entfernt von der Er-
kennungssequenz. Diese Restriktionsendonucleasen spielen
in der Gentechnik praktisch keine Rolle.
(B) Typ-2-Restriktionsendonucleasen:
hydrolysieren die DNA innerhalb der Erkennungssequenz
Spalten sie diese Sequenz symmetrisch, entstehen DNA-
Fragmente mit „glatten Enden“ (z.B. Hae III, Sma I, Dnp I).
Spalten die Enzyme die Erkennungssequenz asymmetrisch,
entstehen DNA-Fragmente, bei denen entweder das
5‘-Ende über das 3‘-Ende hinausragt (5‘ Überhang, z.B.
EcoR I oder BamH I) oder das 3‘-Ende über das 5‘-Ende
hinausragt (3‘ Überhang, z.B. Kpn I oder Pvu I).
Einige Enzyme spalten nur, wenn eine Base innerhalb der
Erkennungssequenz methyliert ist (z.B. Dpn I).
In der Gentechnik gehören die Typ-2-Restriktionsendo-
nucleasen zu den wichtigsten Restriktionsendonucleasen.
(C) Typ-3-Restrktionsendonucleasen:
hydrolysieren die DNA – ähnlich wie die Typ-1-Restriktions-
endonucleasen – unterhalb (downstream) der Erkennungs-
sequenz. Allerdings liegen die Spaltstellen deutlich näher
an der Erkennungssequenz, als dies bei Typ-1-Restriktions-
endonucleasen der Fall ist.
Genetic Engineering 3 EnzymesAxis of Symmetry in DNA Sequences
Most restriction enzymes bind to specific sequences in DNA (restriction
sites) that are symmetrical about their mid points.
The figure shows the restriction site for the enzyme EcoRI on DNA. The axis
of symmetry does not imply that the enzyme cuts along that axis.
Genetic Engineering 3 EnzymesRestriction Cutting Sites
Depending on the enzyme, the cut can occur in one of three ways, as illustrated in
the figures below.
EcoRI PstI SmaI
Genetic Engineering 3 EnzymesCleavage Example 1: EcoRI Genetic Engineering 3 Enzymes
After cleavage
EcoRI PstI SmaI
5‘Überhang 3‘Überhang Glattes Ende
(blunt end)
Genetic Engineering 3 EnzymesProducts generated by restriction enzymes
COHESIVE ENDS
EcoRI 5’…GAATTC…3’ 5’…G AATTC…3’
3’…CTTAAG…5’ 3’…CTTAA G…5’
PstI 5’…CTGCAG…3’ 5’…CTGCA G…3’
3’…GACGTC…5’ 3’…G ACGTC…5’
BLUNT ENDS
HaeIII 5’…GGCC…3’ 5’…GG CC…3’
3’…CCGG…5’ 3’…CC GG…5’
Genetic Engineering 3 EnzymesProducts generated by restriction enzymes cont. Genetic Engineering 3 Enzymes
Isoschizomers: Some enzymes from different strains recognize the same sequences. These are known as isoschizomers. For example, MboI is an isoschizomer of Sau3A and DpnII in that all three enzymes cleave the site GATC. Other enzymes recognize a subset of another enzyme‘s site. For example, MboI (GATC) will recognize, cleave, and leave the same overhangs as BamHI (GGATCC) and BglII (AGATCT). Genetic Engineering 3 Enzymes
Application of Restriction Endonucleases Type II
¾ Complete and partial digestion of DNA.
¾ Restriction fragment length polymorphism.
¾ Restriction mapping.
¾ Cloning procedure.
Genetic Engineering 3 EnzymesPartial restriction enzyme digestion allows cloning of overlapping fragments
a “contig”
• isolation of ~20 kb fragments provides optimally
sized DNAs for cloning in bacteriophage
• partial digestion with a frequent-cutter (4-base cutter) allows production
of overlapping fragments, since not every site is cut
• overlapping fragments insures that all sequences in the genome are cloned
• overlapping fragments allows larger physical maps to be constructed as
contiguous chromosomal regions (contigs) are put together from
the sequence data
• number of clones needed to fully represent the human genome (3 X 109 bp)
assuming ~20 kb fragments
• theoretical minimum = ~150,000
• 99% probability that every sequence is represented = ~800,000All possible sites:
Results of a partial digestion: = uncut = cut
Genetic Engineering 3 EnzymesRestriction Fragment Length Polymorphisms (RFLP) The assortment of DNA fragments after treatment with an restriction endonuclease would represent a specific "fingerprint" of the particular DNA being digested. Different DNA would not yield the same collection of fragment sizes. Thus, DNA from different sources can be either matched or distinguished based on the assembly of fragments after restriction endonuclease treatment. These are termed "Restriction Fragment Length Polymorphisms", or RFLP's. This simple analysis is used in various aspects of molecular biology as well as a law enforcement and genealogy. For example, genetic variations which distingish individuals also may result in fewer or additional restriction endonuclease recognition sites. Genetic Engineering 3 Enzymes
Use of restriction enzymes in the detection of RFLP
(Restriction Fragment Length Polymorphism)
Some DNA sequence changes that produce a restriction fragment length polymorphism (RFLP).
Genetic Engineering 3 EnzymesDetection of an RFLP by Southern blotting. Genetic Engineering 3 Enzymes
Use of restriction enzymes in restriction mapping Genetic Engineering 3 Enzymes
Restriction mapping. Genetic Engineering 3 Enzymes
Genetic Engineering 3 Enzymes
Use of restriction enzymes in cloning of a recombinant plasmid. Genetic Engineering 3 Enzymes
DNA – modifizierende Enzyme
• Ligasen
• Enzyme, die die Enden modifizieren
- alkalische Phosphatase
- Polynucleotidkinase
- Terminale Transferase
• Polymerasen
- DNA Polymerase
- Klenow-Fragment
- Reverse Transcriptase
• Nucleasen:
- Endonucleasen (Dnase I; S1-Nuclease; Restriktionsendonucleasen)
- Exonucleasen (Bal31; ExoIII)
Genetic Engineering 3 EnzymesDNA ligase ¾ The natural function of this enzyme is DNA repair. It will repair discontinuities in a DNA strand and can be used to ligate blunt or sticky ends. ¾ Ligases catalyze the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini of nucleotides (potentially RNA or DNA depending on the ligase). ¾ In a sense, they are the opposite of restriction endonucleases, but they do not appear to be influenced by the local sequence, per se. ¾ Ligases require either rATP or NAD+ as a cofactor, and this contrasts with restriction endonucleases. Genetic Engineering 3 Enzymes
Action of DNA ligase. Genetic Engineering 3 Enzymes
DNA-Ligasen
¾ katalysieren die Bildung einer Phosphodiester-Bindung zwischen einer 5'-Phosphat-Gruppe
und einer benachbarten 3'-Hydroxyl-Gruppe
¾ Reaktion kann sowohl mit kohäsiven Enden als auch mit blunt-ends (nur T4 Ligase)
durchgeführt werden
Zwei verschiedene Ligasen
¾ Vom Bakteriophagen T4 kodierte Ligase
¾ bakterielle Ligase aus E. coli
¾ Hauptunterschied der beiden Ligasen ist das verwendete Co-Enzym und außerdem die spezielle
Fähigkeit der T4 Ligase blunt-ends zu ligieren.
A: T4-DNA-Ligase
B: E.coli-DNA-Ligase
Genetic Engineering 3 EnzymesDNA Ligasen
Alkalische Phosphatase
• A.P. ist ein Enzym, das vor allem in Leber, Knochen, Dünndarm sowie
Gallenblase und -gang vorkommt. Bei Erkrankungen dieser Organe
(v.a. bei Gallengangverschluß) kann die a. P. im Blut erhöht sein.
(Normalwert: ca. 60- 170 U/l)
Anwendungen der Alkalischen Phosphatase
• Klonierungen, um die Religation von Vektor-Enden zu verhindern
• radioaktive Markierung (von 5'-Enden von DNA z.B. mit radioaktivem Phosphor
in Verbindung mit Kinase)
Genetic Engineering 3 EnzymesAlkalische Phosphatase
Hydrolyse von 5‘ P von DNA und RNA
Entfernen des 5'-Phosphat-Restes:
• bakterielle alkalische Phosphatase (BAP) aus E. coli
• alkalische Phosphatase aus dem Kälberdarm (CIP: calf intestine phosphatase;
10- bis 20-fach höhere spezifische Aktivität als die BAP)
• Zink-Ionen (Zn2+) abhängig
• hinterlassen am 5'-Ende als Folge der Hydrolyse eine 5'-Hydroxyl-Gruppe.
Phosphate an 5'-Überhängen, als auch 5'-eingerückte Phosphate (5'-recessed) hydrolysiert.
Genetic Engineering 3 EnzymesCalf intestinal phosphatase (CIP)
• Catalyzes the removal of 5' phosphate groups from RNA, DNA and ribo- and
deoxyribo- nucleoside triphosphates (e.g. ATP, rATP).
• CIP treated duplex DNA cannot self ligate.
• Hemi-phosphorylated duplexes will be ligated on one strand (the
phosphorylated strand) and remain "nicked" on the other.
Genetic Engineering 3 EnzymesT4-Polynucleotidkinase
• Stammt aus dem Bakteriophagen T4
• Ursprünglich aus mit T4 infizierten E. coli gewonnen; heute liegt rekombinantes
Gen vor (in E. coli exprimiert)
• Phosphorylierung von 5‘OH Enden (durch alkalische Phosphatase generiert)
Anwendungen der T4-Polynucleotidkinase
• Klonierungen von PCR-Produkten (Gebrauch nicht-phosphorylierten Oligos und
dephosphoryliertem Vektor)
• radioaktive Markierung (von 5'-Enden von DNA z.B. mit radioaktivem Phosphor)
Genetic Engineering 3 EnzymesT4 polynucleotide kinase
• Catalyzes the transfer and exchange of a phosphate group from the γ position
of rATP (adenine ribose triphosphate nucleotide) to the 5' hydroxyl terminus of
double stranded and single stranded DNA or RNA, and nucleoside 3'
monophosphates.
• The enzyme will also remove 3' phosphoryl groups.
• Oligonucleotides which are obtained from automated synthesizers lack
a 5' phosphate group, and thus, cannot be ligated to other
polynucleotides.
T4 polynucleotide kinase can be used to phosphorylate the 5' end of such polynucleotides:T4-Polynucleotidkinase
„Forward reaction“
„Exchange reaction“
Genetic Engineering 3 EnzymesA procedures that is used routinely for labelling DNA molecules.
End-labelling using T4 polynucleotide kinase
Genetic Engineering 3 EnzymesTerminale Deoxynukleotidyl DNA Polymerase (Terminale Transferase)
• Polymerisiert Template-unabhängig Nukleotide
• aus Kalbs-Thymus gewonnen
• Verwendung in Klonierungen, besitzt aber auch eine Bedeutung für die
3'-Markierung von DNA
Genetic Engineering 3 EnzymesGenetic Engineering 3 Enzymes
Nucleases
Nucleases are enzymes which act on DNA. Exonucleases are enzymes which
remove nucleotides one at a time from the end of a DNA molecule.
Endonucleases are enzymes which are able to break internal phosphodiester
bonds within a DNA molecule.
Exonucleases
Bal31
Exonuclease III
Endonucleases
Mung Bean Nuclease
DNAse I
S1 nuclease
Genetic Engineering 3 EnzymesExonukleasen
Exonukleasen spielen wie die vorausgehend genannten Enzyme ebenfalls
eine wichtige Rolle für verschiedene Klonierungs-Strategien und andere
Anwendungen. Generell wird zwischen Exonukleasen mit Spezifität für einzel-
strängige und/oder doppelsträngige DNA und mit Spezifität für das 3'-
und/oder 5'-Ende unterschieden.
Bal31 is a typical exonuclease. It removes nucleotides from both ends of a DNA strand.
Genetic Engineering 3 EnzymesBal 31 Nuklease
Obwohl die Bal 31 Nuklease aus Alteromonas espejiana per definitionem eine
Einzelstrang-spezifische Endonuklease ist, kann sie unter entsprechenden
Bedingungen verschiedene Reaktionen katalysieren.
Doppelsträngig-zirkuläre DNA kann von Bal 31 zum einen im Bereich von
Nicks zum anderen ausgehend von transient einzelsträngiger DNA, die durch
Supercoiling entstehen kann, abgebaut werden. Lineare Doppelstrang-DNA
wird ebenfalls ausgehend von Nicks abgebaut, aber auch von den 5'- und 3'-
Enden her. Da der Verdau dabei in beiden Richtungen erfolgt, kommt es zu
einer kontrollierten Verkürzung der DNA, was auch eines der
Hauptanwendungs-Gebiete für dieses Enzym ist. Da die Bal 31 Endonuclease
neben Mg2+ auch Ca2+ für ihre Aktivität benötigt, läßt sie sich durch den
Chelator EGTA, der die Magnesium-Konzentration unverändert läßt, aber
Ca2+ bindet, spezifisch inhibieren.
Genetic Engineering 3 EnzymesExonucleases
Exonuclease III
Exonuclease III degrades only the 3' end of the strand. It can be used to
produce single strands.
Genetic Engineering 3 EnzymesExonuclease III (exo III) Exonuclease III wird eingesetzt, um in Verbindung mit Klenow-Polymerase Strang- spezifische radioaktive Proben zu synthetisieren. Sie wird ferner benutzt, um Einzel-Strang DNA für Sequenzierungen bereitzustellen. Da die Exonuklease III spezifisch für Doppel-Strang-DNA ist, verdaut sie bevorzugt DNA mit 5'-Überhängen von der zurückgesetzten 3'-OH-Gruppe her. 3'-Überhänge sind praktisch resistent gegenüber Verdau. Diesen Mechanismus kann man sich für spezielle Sequenzier- Strategien nutzbar machen oder um definierte Verkürzungen von DNA-Fragmenten von nur einer Seite her (erase-a-base System) zu ermöglichen. Bei Einsatz der Exonuklease III ist darauf zu achten, daß ihre Abbaurate abhängig ist von der Basenzusammensetzung der zu verdauenden DNA, weshalb unterschiedliche Substrate mit verschiedenen Geschwindigkeiten verdaut werden können (C >> A T >> G). Genetic Engineering 3 Enzymes
Bal31 ExoIII
Nuclease BAL-31
• This is an exonuclease (starts at the termini and works inward) which will
degrade both 3' and 5' termini of double stranded DNA. It will not make internal
cleavages ("nicks"), however, it will degrade the ends of DNA at existing
internal "nicks" (which create both 3' and 5' termini).
• The degradation of termini is not coordinated, meaning that the product is not
100% blunt ended (even though the original duplex may have been blunt
ended).
• Such "ragged" ends can be made blunt by filling in and chewing back by a
suitable polymerase (e.g. T4 DNA polymerase). The unit definition is 1 unit is
amount of enzyme required to remove 200 base pairs from each end of duplex
DNA in 10 minutes at 30 °C.Exonuclease III
• Catalyzes the stepwise removal of nucleotides from the 3' hydroxyl termini of
duplex DNA.
• The enzyme will attack the 3' hydroxyl at duplex DNA with blunt ends, with 5'
overhangs, or with internal "nicks".
• Since duplex DNA is required, the enzyme will not digest the 3' end of duplex
DNA where the termini are 3' overhangs.
Genetic Engineering 3 EnzymesEndonucleases
S1 nuclease
S1 nuclease acts on ssDNA.
S1 Nuclease cleaves phosphodiester bonds at
any point within an ssDNA molecule.
DNase I
DNase 1 acts on ssDNA or dsDNA. It will cleave all DNA, eventually into single nucleotides.
DNase I can cleave any phosphodiester bond in
ssDNA or dsDNA.
Genetic Engineering 3 EnzymesS1 Nuklease Die S1 Nuklease aus Aspergillus oryzae ist eine hoch-spezifische Einzelstrang- Endonuklease. Sie wird unter anderem in Verbindung mit Exonuklease III zum kontrollierten Verkürzen von DNA eingesetzt (erase-a-base System). Ebenso wie Bal 31 Endonuklease kann sie doppelsträngige DNA im Bereich von Nicks verdauen. Ihre Hauptanwendung ist allerdings die Entfernung von Überhängen an DNA, da sie in blunt-ends praktisch nicht hineinverdaut und DNA so klonierbar machen kann. Mung Bohnen Nuklease (Mung Bean) Die Spezifität der Mung Bean Nuklease ist vergleichbar der der S 1 Nuklease und sie wird entsprechend für ähnliche Zwecke eingesetzt. Ihr Vorteil besteht darin, daß sie wesentlich präziser verdaut, als die S 1 Nuklease, weshalb sie für die Bestimmung von Transkript-Anfängen und -Enden deutliche Vorzüge hat. Sie spaltet im Gegensatz zur S 1 Nuklease nicht im Bereich von Nicks. Genetic Engineering 3 Enzymes
Mung Bean Nuclease (isolated from mung bean sprouts)
• A single strand specific DNA and RNA endonuclease which will degrade single
strand extensions from the ends of DNA and RNA leaving blunt ends.
• The single strand extensions can be either 5' or 3' extensions - both are
removed and a blunt duplex is left.
Genetic Engineering 3 EnzymesDeoxyribonuclease I (DNase I) Die DNase I, die aus dem Pankreas von Rindern stammt, ist eine Endonuklease mit Spezifität für Doppelstrang-DNA. Unter ihrer Wirkung entstehen Oligonukleotide mit 3'-Hydroxyl-Gruppen. Das Enzym zeigt in Abhängigkeit von den Inkubations- Bedingungen unterschiedliche Spezifitäten. In Gegenwart von Mg2+ produziert die DNase I Nicks in doppelsträngiger DNA, während sie in Gegenwart von Mn2+ Doppelstrangbrüche verursacht. Während sie früher in erster Linie für die radioaktive Markierung über die sogenannte Nick- Translation eingesetzt wurde, findet sie heute neben ihrer Hauptaufgabe RNA- Präparationen DNA-frei zu bekommen, Verwendung für die Bestimmung von DNA- Protein-Wechsel-Wirkungen.
Deoxyribonuclease I (DNAse I) from Bovine pancrease
• This enzyme hydrolyzes duplex or single DNA strands preferentially at the
phosphodiester bonds 5' to pyrimidine nucleotides
• In the presence of Mg2+ ion, DNAse I attacks each strand independently
and produces nicks in a random fashion (useful for nick-translation)
• In the presence of Mn2+ ion DNAse I cleaves both strands of DNA at
approximately the same position (but leaving "ragged" ends)
Genetic Engineering 3 EnzymesDNase I and nick translation
DNA polymerases
• E. coli DNA polymerase I
• Klenow-Fragment
• T4-DNA polymerase
• Terminal Transferase
• Reverse Transcriptase
• Taq polymerase
Genetic Engineering 3 EnzymesDNA Polymerases
A wide variety of polymerases have been characterized and are commercially
available. All DNA polymerases share two general characteristics:
1. They add nucleotides to the 3'-OH end of a primer
2. The order of the nucleotides in the nascent polynucleotide is template
directed
¾ In addition to the 5'->3' polymerase activity, polymerases can contain
exonuclease activity. This exonuclease activity can proceed either in the 5'-
>3'direction, or in the 3'->5' direction.DNA-abhängige DNA-Polymerasen Neben den Nukleasen finden eine Reihe von Polymerasen Anwendung für die Manipulation von DNA-Fragmenten, die zur Klonierung kommen sollen, und die wir daher nachfolgend kurz besprechen wollen. Allen DNA-Polymerasen gemeinsam ist ihre Fähigkeit, Deoxyribonukleotide an ein freies 3'-Hydroxyl-Ende anzuhängen. Dabei erfolgt die Synthese stets nur von 5' nach 3‘. Die meisten Polymerasen besitzen neben der synthetisierenden Aktivität auch 3' > 5' Exonuklease-Aktivitäten. Diese Aktivität, die im Organismus Korrektur-Funktion hat, kommt insbesondere dann ins Spiel, wenn die Konzentration an freien Nukleotiden für die Polymerase-Reaktion einen kritischen Wert unterschreitet. Sind genügend Nukleotide vorhanden, überwiegt allerdings die Polymerase-Reaktion. Einige der im gentechnischen Bereich eingesetzten Polymerasen (z.B. E. coli DNA Polymerase I) besitzen zudem noch eine 5' -> 3' Exonuklease - Aktivität, durch die DNA vom 5'-Ende her unter Abspaltung von Mono- oder Oligonukleotiden verdaut werden kann. Genetic Engineering 3 Enzymes
¾ Exonuclease activity in the 3'->5' direction allows the polymerase to correct a mistake if it incorporates an incorrect nucleotide (so called "error correction activity"). It can also slowly degrade the 3' end of the primer. ¾ Exonuclease activity in the 5'->3' direction will allow it to degrade any other hybridized primer it may encounter. Without 5'->3' exonuclease activity, obstructing primers may or may not be physically deplaced, depending on the polymerase being used. ¾ Different polymerases have differing error rates of misincorporation, and different rates of polymerization.
The DNA polymerase reaction.
Each incoming dNTP is positioned
by base-pairing with the appropriate
template nucleotide, and a phospho-
diester bond is created by nucleophylic
attack of the primer-strand 3' hydroxyl
group onto the alpha phosphate of the
incoming dNTP.
1. 5'-to-3' DNA Polymerase activity
2. 3'-to-5' exonuclease (Proofreading activity)
3. 5'-to-3' exonuclease (Nick translation activity)
Genetic Engineering 3 EnzymesSynthesis of DNA Genetic Engineering 3 Enzymes
Some examples of DNA polymerases
Enzyme Template Primer Other activities Other features
E. coli DNA polI DNA DNA/RNA 3'-5' exo, 5'-3' exo monomeric
E. coli DNA polI DNA DNA/RNA 3'-5' exo C-terminal fragment
(Klenow fragment)
E. coli DNA pol III DNA DNA/RNA 3'-5' exo (on a multimeric structure
separate subunit)
Taq pol DNA DNA/RNA extendase (adds thermostable, used in
3'-A overhangs) PCR
reverse transcriptase DNA/RNA DNA/RNA (ribonuclease H) used to make cDNA
terminal transferase none DNA will synthesize DNA
required in non-templated
reactionUses of polymerases
The various activities of the different polymerases lend them to a variety of
applications.
For example, restriction endonucleases can yield fragments of DNA with either 3' or 5'
nucleotide "overhangs".
• In the case of 5' overhangs, the 5'->3' polymerase activity can fill these in to make blunt
ends.
• In the case of 3' overhangs, the 3'->5' exonuclease activity present in some polymerases
(especially T4 DNA polymerase) can "chew back" these ends to also make blunt-ended
DNA fragments."Nick-translation"
This method is used to obtain highly radiolabeled single strand DNA fragments,
which makes use of 5'->3' exonuclease activity present in some polymerases (E. coli
DNA polymerase I, for example).
• In this method a DNA duplex of interest is "nicked" (i.e. one of the strands is
cut; see DNAse I).
• Then DNA pol I is added along with radiolabeled nucleotides. The 5'->3'
exonuclease activity chews away the 5' end at the "nick" site and the
polymerase activity incorporates the radiolabeled nucleotides. The resulting
polynucleotide will be highly radiolabeled and will hybridize to the DNA
sequence of interest.Large (Klenow) Fragment of E. coli DNA Polymerase I The 5' -> 3' exonuclease activity of E. coli's DNA polymerase I makes it unsuitable for many applications. However, this pesky enzymatic activity can readily be removed from the holoenzyme. Exposure of DNA polymerase I to the protease subtilisin cleaves the molecule into a small fragment, which retains the 5' -> 3' exonuclease activity, and a large piece called Klenow fragment. The large or Klenow fragment of DNA polymerase I has DNA polymerase and 3' -> 5' exonuclease activities, and is widely used in molecular biology. Genetic Engineering 3 Enzymes
Klenow fragment is useful for several tasks: Synthesis of double-stranded DNA from single-stranded templates: The function of DNA polymerases is to synthesize complementary strands during DANN replication. Performing that task in the lab is integral to such processes as synthesizing the second strand DNA in cDNA cloning and generating radioactive probes for hybridization reactions. DNA polymerases require a primer to provide a free 3' hydroxyl group for initiation of synthesis. The primers used for most in vitro polymerization reactions are single-stranded DNAs, typically 6 to 20 bases in length, called oligonucleotides. The oligonucleotides must be complementary to some section of template DNA. To use Klenow to synthesize a complementary strand of DNA, one simply mixes single-stranded template (usually denatured double-standed DNA), primers and the enzyme in the presence of an appropriate buffer (most restriction enzyme buffers work well). The reaction proceeds are depicted below: Strand displacement! One item of some significance in the above reaction is that as Klenow proceeds, it can displace primers downstream and continue synthesizing new DNA.
Klenow fragment Filling in recessed 3' ends of DNA fragments: A "fill-in" reaction is used to create blunt ends on fragments created by cleavage with restriction enzymes that leave 5' overhangs. This reaction is conceptually identical to the one described above, but with a huge primer and a very short segment of single-stranded template. Digesting away protruding 3' overhangs: This is another method for producing blunt ends on DNA, in this with ends generated from restriction enzymes that cleave to produce 3' overhangs. The 3' -> 5' exonuclease activity of Klenow will digest away the protruding overhang. Removal of nucleotides from the 3' ends will continue, but, in the presence of nucleotides, the polymerase activity will balance the exonuclease activity, yielding blunt ends. This reaction is more efficienty conducted with T4 DNA polymerase, which has much more potent exonuclease activity.
Klenow-Fragment der DNA-Polymerase I Das Klenow-Fragement der Polymerase I von E. coli ist ein Fragment der Polymerase, das die Polymerase-Funktion und die 3' -> 5' - Exonuklease-Aktivität der Polymerase besitzt, nicht jedoch die 5' - 3' -Exonuklease-Aktivität, die im ursprünglichen Enzym im N-Terminus des Proteins lokalisiert ist. Ursprünglich wurde das Klenow-Fragment durch proteolytischen Verdau von E. coli DNA Polymerase I gewonnen. Mittlerweile existiert allerdings ein geklontes Gen, das die terminalen 323 Aminosäuren nicht mehr kodiert. Das Klenow-Fragment wird in erster Linie eingesetzt, wenn 5'-Überhänge im Rahmen eines Klonierungs-Experimentes aufgefüllt werden sollen, oder durch den Einbau radioaktiv markierter Nukleotide Sonden erstellt werden sollen. Der Einsatz des Klenow- Fragmentes zur Herstellung radioaktiv-markierter DNA erfolgt in erster Linie in Verbindung mit Zufalls-bindenden Hexanukleotiden. Dabei ist insbesondere die Fähigkeit des Fragmentes zum sogenannten strand-displacement von Bedeutung. Die Verwendung des Klenow-Fragmentes für die Sequenzierung von DNA ist etwas aus der Mode gekommen, da für diesen Zweck Polymerasen mit höherer Prozessivität eindeutige Vorteile haben, wie z.B. T7 Polymerase. Genetic Engineering 3 Enzymes
The Principle of Random Primed Labeling A procedures that is used routinely for labelling DNA molecules. Random primed labelling using the large fragment of DNA polymerase I (Klenow fragment).
T4 DNA Polymerase Die aus dem Bakteriophagen T4 stammende Polymerase weist ebenso wie das Klenow- Fragment der E. coli Polymerase I keine 5' -> 3' Exonuklease-Aktivität auf. Neben seiner Polymerase-Aktivität besitzt es eine sehr aktive 3' -> 5' Exonuklease-Aktivität. Genetic Engineering 3 Enzymes
T4-DNA-Polymerase – Markierung von DNA Fragmenten Genetic Engineering 3 Enzymes
T7 DNA Polymerase The DNA polymerase of T7 bacteriophage has DNA polymerase and 3' -> 5‘ exonuclease activities, but lacks a 5' -> 3' exonuclease domain. It is thus very similar in activity to Klenow fragment and T4 DNA polymerase. The claim to fame for T7 DNA polymerase is it's processivity. That is to say, the average length of DNA synthesized before the enzyme dissociates from the template is considerably greater than for other enzymes. Due to this talent, the principle use of T7 DNA polymerase is in DNA sequencing by the chain termination technique. T7 DNA polymerase can be chemically-treated or genetically engineered to abolish it's 3' -> 5‚ exonuclease activity. These forms of the enzyme are marketed under the name Sequenase and Sequenase 2.0, and are widely used for DNA sequencing reactions. Genetic Engineering 3 Enzymes
T7 Polymerase Dieses ursprünglich aus mit T7 Bakteriophagen infizierten E. coli gewonnene Enzym zeichnet sich durch eine enorm hohe Prozessivität in Gegenwart von Thioredoxin aus, wodurch es in der Lage ist in vitro Tausende von Nukleotiden ausgehend von einem Start aus zu polymerisieren. Da der Polymerase/Thioredoxin-Komplex kaum durch Sekundär-Strukturen der Template-DNA gestört wird, der Komplex aber eine hohe 3' -> 5' Exonuklease-Aktivität aufweist, wurde eine modifizierte T7 DNA Polymerase erzeugt, die diese Aktivität nicht mehr besitzt (28 Aminosäuren der Exonuklease-Domäne wurden deletiert). Hierdurch konnte die Prozessivität weiter gesteigert werden, was das modifizierte Enzym zu einem Mittel der Wahl bei DNA- Sequenzierungen nach der Dideoxy-Methode macht. (chapter sequencing) Genetic Engineering 3 Enzymes
Reverse Transcriptases Reverse transcriptase is a common name for an enzyme that functions as a RNA-dependent DNA polymerase. They are encoded by retroviruses, where they copy the viral RNA genome into DNA prior to ist integration into host cells. Reverse transcriptases have two activities: DNA polymerase activity: In the retroviral life cycle, reverse transcriptase copies only RNA, but, as used in the laboratory, it will transcribe both single-stranded RNA and single-stranded DNA templates with essentially equivalent efficiency. In both cases, an RNA or DNA primer is required to initiate synthesis. RNase H activity: RNase H is a ribonuclease that degrades the RNA from RNA-DANN hybrids, such as are formed during reverse transcription of an RNA template. This enzyme functions as both an endonuclease and exonuclease in hydrolyzing its target. Genetic Engineering 3 Enzymes
The life cycle of a retrovirus depends on reverse transcriptase
retrovirus 2. the capsid is uncoated, releasing genomic
RNA and reverse transcriptase
3. reverse transcriptase
1. virus enters cell makes a DNA copy
and looses envelope
4. then copies the DNA strand to
make it double-stranded DNA,
6. it is translated into viral proteins, removing the RNA with RNase H
and assembled into new
virus particles
5. the DNA is then integrated
into the host cell genome
where it is transcribed by
new viruses host RNA polymerase II
Genetic Engineering 3 EnzymesReverse transcriptase
All retroviruses have a reverse transcriptase, but the enzymes that are available
commercially are derived from one of two retroviruses, either by purification from the
virus or expression in E. coli:
- Moloney murine leukemia virus: a single polypeptide
- Avian myeloblastosis virus: composed of two peptide chains
Both enzymes have the same fundamental activities, but differ in a number of
characteristics, including temperature and pH optima. Most importantly, the murine
leukemia virus enzyme has very weak RNase H activity compared to the avian
myeloblastosis enzyme, which makes it the clear choice when trying to synthesize
complementary DNAs for long messenger RNAs.
Genetic Engineering 3 EnzymesReverse transcriptase Reverse transcriptase is used to copy RNA into DNA. This task is integral to cloning complementary DNAs (cDNAs), which are DNA copies of mature messenger RNAs. The technique is usually initiated by mixing Short (12-18 base) polymers of thymidine (oligo dT) with messenger RNA such that They anneal to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the oligo dT as a primer to synthesize so-called first-strand cDNA. Another common use for reverse transcriptase is to generate DNA copies of RNAs prior to amplifying that DNA by polymerase chain reaction (PCR). Reverse transcription PCR, usually called simply RT-PCR, is a useful tool for such things as cloning cDNAs, diagnosing microbial diseases rapidly and a myriad of other applications. In most cases, standard preparations of reverse transcriptase are used for RT-PCR, but mutated forms with relatively high thermal stability have been developed to facilitate the process.
Reverse Transkriptase Genetic Engineering 3 Enzymes
Purification of PolyA-containing mRNA A mixture of total eukaryotic cellular RNA molecules (rRNA, mRNA, and tRNA) is hybridized to oligo dT chemically attached to a support matrix (green column). PolyA-containing messenger RNA, mRNA (blue), is retained on the column while the ribosomal RNA, rRNA (red), and transfer RNA, tRNA (green), flow through. Hydrogen bonding is then disrupted, allowing the collection of mRNA free of rRNA and tRNA Genetic Engineering 3 Enzymes
cDNA Synthesis Eukaryotic mRNA (blue) has a polyA tail. A DNA copy (cDNA) can be made with reverse transcriptase using oligo dT as a primer (red). After the RNA is destroyed by NaOH treatment, double-stranded DNA can be made with DNA polymerase using random hexamer primers (green). The total population of cDNAs can be cloned as a cDNA library, or a particular cDNA can be amplified by PCR using gene- specific primers. Genetic Engineering 3 Enzymes
The synthesis of cDNA. Genetic Engineering 3 Enzymes
Thermostable DNA Polymerases The original report of this enzyme, purified from the hot springs bacterium Thermus aquaticus, was published in 1976. Roughly 10 years later, the polymerase chain reaction was developed and shortly thereafter "Taq" became a household word in molecular biology circles. Currently, the world market for Taq polymerase is in the hundreds of millions of dollars each year. The thermophilic DNA polymerases, like other DNA polymerases, catalyze template- directed synthesis of DNA from nucleotide triphosphates. A primer having a free 3' hydroxyl is required to initiate synthesis and magnesium ion is necessary. In general, they have maximal catalytic activity at 75 to 80°C, and substantially reduced activites at lower temperatures. At 37°C, Taq polymerase has only about 10% of its maximal activity. Genetic Engineering 3 Enzymes
In addition to Taq DNA polymerase, several other thermostable DNA polymerases have been isolated and expressed from cloned genes. Three of the most-used polymerases are described in the following table: Genetic Engineering 3 Enzymes
Thermostable polymerases and error rates One of the most discussed characteristics of thermostable polymerases is their error rate. Error rates are measured using several different assays, and as a result, estimates of error rate vary, particularly when the assays are performed by different labs. As would be expected from first principles, polymerases lacking 3'->5' exonuclease activity generally have higher error rates than the polymerases with exonuclease activity. The total error rate of Taq polymerase has been variously reported between 1 x 10-4 to 2 x 10-5 errors per base pair. Pfu polymerase appears to have the lowest error rate at roughly 1.5 x 10-6 error per base pair, and Vent is probably intermediate between Taq and Pfu. Genetic Engineering 3 Enzymes
Objectives chapter 3 (enzymes):
• Be able to draw the methylated bases
• Understand how the restriction/modification system works and its function in bacteria.
Be able to explain why methylases are monomeric proteins and associated restriction
endonucleases are dimeric (i.e. as relates to protection of host DNA during replication)
• Be familiar with the chart of restriction endonucleases; the name, recognition sequence
and cleavage sites (do not learn recognition sequences). Understand what is meant by 3'
overhang, 5' overhang, and blunt cuts.
• Understand the relationship between size of recognition sequence and frequency of
occurance
• Understand how the different enzymes work
• Understand the general characteristics of polymerases, including exonuclease activities
• Given different examples of oligonucleotides be able to predict the results of treatment
with different polymerases, nucleases, kinases, phosphatases
• Be able to suggest methods of treating DNA in order to be able to achieve an
experimental result. For example: given two DNA fragments, one containing 3' overhangs,
the other 5' overhangs, how could we treat these so that we could ligate the fragments
together?
• Understand methods of labeling DNA (end labeling; nick-translation; random primed
labeling)
• Know how mRNA is copied into cDNA
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