Enzymes for manipulating DNA

1
Enzymes for manipulating DNA

• Buffers and solution conditions
• I. DNA polymerases
• III. Kinase and alkaline phosphatase
• IV. Nucleases
• V. Topoisomerase
• Course Readings 19 and 20

2

• Buffers are crucial for activity of enzymes!
• Ideal biochemical buffers
• pKa between 6 and 8
• Chemically inert
• Polar (soluble and not membrane permeable)
• Non-toxic
• Inexpensive
• Salt and temperature indifferent
• Tris pKa is 8.0
• Tris(hydroxymethyl)aminomethane (THAM) the free
  base for (pH 7.5-8.5)
• Tris-HCl the acidic form (for pH 7-8)

3

• Tris is widely used, but it isnt perfect
• Buffering is weak below pH 7.5 and above pH 9.0
• pH must be measured using a special pH meter
  electrode
• Toxic to many types of mammalian cell cultures
• Tris solution pH changes with temperature! Drops
  0.03 pH units for each degree C increase
• Tris solution pH changes with concentration!
  Example 10mM Tris pH 7.9, 100mM Tris pH 8.0
• Below pH 7.5, use a Good buffer HEPES,
  Tricine, BES, MOPS, MES

4

• Enzyme reaction buffers
• Buffer Tris, HEPES, etc.
• Salt NaCl, KCl, PO4-, etc.--stabilizes protein
  structure, facilitates protein-DNA interactions
• Divalent metal ions Mg2, Ca2, Zn2,
  etc.--often required for enzyme activity
• Glycerol (for storage)--stabilizes protein
  structure
• EDTA chelates (removes) divalent
  cations--important especially for storage, if
  your enzyme is especially sensitive to metal
  ion-dependent proteases
• Beta mercaptoethanol or dithiothreitol reducing
  agents that prevent illegitimate disulfide bond
  formation
• Non-specific protein Bovine serum albumin (BSA)
• Other cofactors, eg. ATP, NADH

5
DNA polymerases--making copies, adding labels, or
fixing DNA

• E. coli DNA polymerase I --the classic DNA
  polymerase
• Moderately processive polymerase
• 3'-gt5' proof-reading exonuclease
• 5'-gt3' strand-displacing (nick-translating)
  exonuclease
• Used mostly for labelling DNA molecules by nick
  translation. For other purposes, the Klenow
  fragment is usually preferred

6
DNA polymerases

• Klenow fragment --the C-terminal 70 of E. coli
  DNA polymerase I originally prepared as a
  proteolytic fragment (discovered by Klenow) now
  cloned
• Lacks the 5'-gt3' exonuclease activity
• Uses include
• Labeling DNA termini by filling in the cohesive
  ends generated by certain restriction enzymes
• generation of blunt ends
• DNA sequencing

7
(No Transcript)
8
A way of making blunt ended DNA (repair after
mechanical fragmentation)
9
A way of radiolabeling DNA
10
DNA polymerases

• Native T7 DNA polymerase --highly processive,
  with highly active 3'-gt5' exonuclease
• Useful for extensive DNA synthesis on long,
  single-stranded (e.g. M13) templates
• Useful for labeling DNA termini and for
  converting protruding ends to blunt ends
• Modified T7 polymerase (Sequenase) --lack of both
  3'-gt5' exonuclease and 5'-gt3' exonuclease
• Ideal for sequencing, due to high processivity
• Efficiently incorporates dNTPs at low
  concentrations, making it ideal for labeling DNA

11
DNA polymerases

• Reverse transcriptase
• RNA-dependent DNA polymerase
• Essential for making cDNA copies of RNA
  transcripts
• Cloning intron-less genes
• Quantitation of RNA

12
Reverse transcriptase The Km for dNTPs is very
high (relatively non-processive) Makes a DNA
copy of RNA or DNA -- but -- The self-primed
second strand synthesis is inefficient Second-st
rand cDNA synthesis is usually done with DNA
polymerase and a primer
13
How RT works
14
cDNA library construction using reverse
transcriptase
cDNA Library Construction Kit (Clontech)
15

• Priming reverse transcriptase
• General RNA amplification
• Oligo(dT)12-18
• Random sequence oligonucleotides
• Specific mRNA
• Single oligonucleotide sequence complementary to
  your mRNA
• NOTE Reverse transcriptase is error-prone (about
  1/500 bp is mutated)

16
Terminal transferase

• template-independent DNA polymerase
• Incorporates dNTPs onto the 3' ends of DNA chains
• Useful for adding homopolymeric tails or single
  nucleotides (can be labelled) to the 3' ends of
  DNA strands (make DNA fragments more easily
  clonable)

17
T4 polynucleotide kinase

• Transfers gamma phosphate of ATP to the 5 end of
  polynucleotides
• Useful for preparing DNA fragments for ligation
  (if they lack 5 phosphates)
• Useful for radiolabelling DNA fragments using
  gamma 32P ATP as a phosphate donor

18
alkaline phosphatase

• Catalyzes removal of 5 (and 3) phosphates from
  polynucleotides
• Useful for treating restricted vector DNA
  sequences prior to ligation reactions, prevents
  religation of vector in the absence of insert DNA
• Lack of vector 5 phosphates may inhibit
  transformation efficiency? Use only when
  absolutely necessary

19
Nucleases

• Exonucleases
• Remove nucleotides one at a time from a DNA
  molecule
• Endonucleases
• Break phosphodiester bonds within a DNA molecule
• Include restriction enzymes

20
Exonucleases

• Bal 31
• Double-stranded exonuclease, operates in a
  time-dependent manner
• Degrades both 5 and 3 ends of DNA
• Useful for generating deletion sets, get bigger
  deletions with longer incubations

21
Exonucleases

• Exonuclease III--double-stranded DNA
• 3-5 exonuclease activity
• 3 overhangs resistant to activity, can use this
  property to generate nested deletions from one
  end of a piece of DNA (use S1 nuclease to degrade
  other strand of DNA)

22
Exonucleases

• Exonuclease I
• 3-5 exonuclease
• Works only on single-stranded DNA
• Useful for removing unextended primers from PCR
  reactions or other primer extension reactions

23
Endonucleases

• Dnase I
• Cleaves double-stranded DNA randomly (also
  cleaves single-stranded DNA)
• Mn both strands of DNA cut
• Mg single strands nicked
• Very useful for defining binding sites for DNA
  binding proteins

24
DNAse I footprinting
Calibrate the nicking 1 hit per DNA molecule
25
Drosophila heat-shock factor
DNAse I footprintingGel following footprinting
reaction
0
Sites for interaction of HSF with DNA
26
Topoisomerase
Function A restriction enzyme and ligase--all in
one altering the linking number in coiled,
constrained (supercoiled) DNA--relaxing DNA
twisting during replication Model for function
http//mcb.berkeley.edu/labs/berger/structures.ht
mlmodeling
27
Cloning with topoisomerase
28
Topoisomerase

• Topoisomerase catalyzed ligation is EXTREMELY
  efficient (gt85 of resulting plasmids are
  recombinant)--excellent for library constructions
• Can be used to clone blunt ended DNA (PCR
  products, restriction digests), T-overhang PCR
  products (from Taq polymerase), and directional
  clones
• You have to use their plasmid vectors (ie. forget
  about using your favorite lab plasmid unless you
  know how to covalently attach topoisomerase)

29
Enzymes for manipulating DNA

• Buffers and solution conditions
• I. DNA polymerases
• III. Kinase and alkaline phosphatase
• IV. Nucleases
• V. Topoisomerase
• Course Readings 19 and 20

30
Cutting and pasting DNA

1. Restriction and modification systems
2. Recognition and cleavage of DNA by restriction
   endonucleases (REases)
3. Joining (ligating) DNA molecules
4. Cloning techniques

31
Discovery of restriction/modification
EOP efficiency of plating (a measure of phage
virulence) bacteriophage E. coli K
has R/M system E. coli C has no M system
32
Cautions for cloning in E.coli
Strains with methylases (dam or dcm) produce
methylated DNA--difficult to cleave with certain
enzymes, hard to transform some strains
Strains with restriction systems intact will
restrict DNA coming from a host lacking
methylases, or from a host with specific types of
methylations Best bet is to delete the
restriction systems, but not all cloning strains
have this deletion
33
Types of endonucleases

• Type I multisubunit proteins that function as a
  single protein complex, usually contain two R
  subunits,two M subunits and one S subunit
• Type II recognize specific DNA sequences and
  cleave at constant positions at or close to that
  sequence to produce 5-phosphates and
  3-hydroxyls. Most useful in cloning!!
• Type III composed of two genes (mod and res)
  encoding protein subunits that function either in
  DNA recognition and modification (Mod) or
  restriction (Res)
• Type IV one or two genes encoding proteins that
  cleave only modified DNA, including methylated,
  hydroxymethylated and glucosyl-hydroxymethylated
  bases

34
Mode of action of type II REases
EcoRI
5 ... GA A T T C ... 3 3 ... C T T A AG ...
5
EcoRI
5 ... G 3 5 A A T T C ... 3 3 ... C
T T A A 5 3 G ... 5
35
Example recognition sequences for REases
4-cutters AluI 5 ... AGCT ... 3 blunt
ends MspI 5 ... CCGG ... 3 5 overhang
(2 bp) 6-cutters PvuII 5 ... CAGCTG ...
3 blunt ends KpnI 5 ... GGTACC ... 3 3
overhang (4 bp) 8-cutters NotI 5 ...
GCGGCCGC ... 3 5 overhang (4 bp) Unusual
sites MwoI 5 ... GCNNNNNNNGC ... 3 3
overhang 3 ... CGNNNNNNNCG ... 5 (3 bp)
36
How often does REase cut my sequence?

• Known sequence scan for sites by computer (eg.
  at www.rebase.neb.com)
• Unknown sequence hypothetical calculations
• 4 cutter site occurs randomly every 44 (256)
  base pairs
• 6 cutter every 46 (4096) bp
• 8 cutter every 48 (65536) bp
• But sequences are not distributed randomly (table
  3.4)
• Sequence context effects
• Some sites are preferred over others by enzyme

37
The ligation reaction

• Biological function of ligases
• Lagging strand DNA synthesis
• genetic recombination
• DNA repair

38
Behavior of cohesive ends (overhangs)
39
Cloning techniques

• A) Modify the ends of the DNAs to make foreign
  DNA sequences more ligate-able
• B) Directional cloning (generate easily cloned
  PCR fragments)
• C) Treat the vector DNA with alkaline phosphatase
  to improve the efficiency of ligation of foreign
  DNA versus vector recircularization

40
Creating a recombinant DNA molecule
Plasmid vector a cloning vehicle it can
replicate itself in a bacterial host and contains
a means for selection (eg. antibiotic resistance)
41

• Ligation efficiency depends on the DNA ends in
  the reaction
• Complementary sticky ends
• Ligation is efficient
• annealing of complementary overhangs brings 5P
  and 3OH into close proximity
• Blunt ends
• Ligation is inefficient
• need high concentrations of ligase and DNA
• molecular crowding reagents (like PEG 8000)
  improve intermolecular ligation, then dilute to
  promote intramolecular ligation
• Follow the manufacturers instructions

42
Cloning foreign DNA by adding linkers
(your DNA molecule should not have EcoRI sites in
this case)
43
Cloning foreign DNA by adding adaptors
The advantage of this is you do not need to treat
the adaptor-modified DNA with restriction enzyme
44
Terminal transferase to add polynucleotide tails
to foreign DNA and vector DNA
Foreign DNA
Vector DNA
dTTP
45
Cloning Taq PCR products

• Taq PCR products have a 3 A overhang
• Prepare vector to have a 3 T overhang

HphI leaves T overhangs
46
Directional cloning
47
Directional cloning
This guarantees the orientation of your DNA
fragment
48
Easy cloning PCR products
Design PCR primers with built in restriction
sites (check amplified sequence for those sites
first!)
Ready for directional cloning
49
Utility of alkaline phosphatase in ligation
Chances of getting recombinant product are
improved
50
Cutting and pasting DNA

1. Restriction and modification systems
2. Recognition and cleavage of DNA by restriction
   endonucleases (REases)
3. Joining (ligating) DNA molecules
4. Cloning techniques

51

• Mobilizing DNA vectors for propagation in E.
  coli
• Plasmids
• Bacteriophage
• M13
• Lambda
• Cosmids and BACs

52
Plasmids and transformation

• Properties of plasmids
• Plasmids as cloning vehicles (vectors)
• Ligation and transformation, and identification
  of recombinant plasmids
• Course Readings 21 (plasmids) and 22
  (antibiotic selection)

53
Plasmids

• Extrachromosomal, double-stranded, usually
  circular, supercoiled DNA molecules
• Found in many bacterial species
• Replicate and are inherited independently of the
  bacterial chromosome
• Maintain copy number in cell through an origin of
  replication (replicon)
• Usually have genes coding for enzymes that
  provide benefits for the host bacterium, eg.
  antibiotic resistance

54
a generic, minimal plasmid
restriction site for cloning
antibiotic resistance
pBi430/530 1500 base pairs (a manageable size
origin of replication
55
Replicon -- how the plasmid replicates

• Governs replication of plasmid and number of
  plasmid copies per cell (copy number)
• A replicon includes
• origin of replication (ori a site on the DNA)
• associated factors
• gt 30 different replicons known, but most plasmids
  used today have pMB1 (or the close relative
  colE1) replicon

56
pMB1/colE1 replication mechanism
1
Deletion of Rop or mutation of RNA II cause
increases in replication and copy number
2
3
4
57
Common plasmids and their stats
PLASMID REPLICON COPY
pBR322 pMB1 15-20
pUC Modified form of pMB1 (RNAII mutation) 500-700
pACYC p15A 18-22
pSC101 pSC101 about 5
58
Plasmid copy number

• High copy number plasmids
• Workhorses of molecular cloning
• Used for almost all routine manipulation of small
  (lt15 kb) recombinant DNAs
• Low copy number plasmids
• For genes that are lethal or unstable in high
  copy number plasmids
• For constructing Bacterial Artificial Chromosomes
  (BACs) that can propagate large (gt100 kb)
  recombinant DNAs

59
Plasmid maintenance

• Plasmids contain selectable markers genes
  carried by the plasmid that confer functions
  required for host survival
• Selection only those cells with the plasmid will
  survive
• Allows transformation (a rare event) to be
  feasible
• A way to keep cells from losing plasmids that may
  otherwise confer a selective disadvantage

60
Antibiotic resistance genes

• Beta lactamase (bla) breaks down ampicillin and
  carbenicillin (inhibitors of cell wall
  synthesis). Cells carrying this gene are often
  termed ampr
• CAUTION Over time beta-lactamase is secreted
  into the medium where it breaks down the
  antibiotic and depletes it. Eventually this
  allows the growth of ampicillin/ carbenicillin
  sensitive cells, defeating the selection

61
Antibiotic resistance genes

• Chloramphenicol acetyl transferase (CAT)
  inactivates chloramphenicol (cm), which normally
  inhibits peptidyl transferase activity of the
  ribosome (no protein synthesis dead cell)
• Another use for cm
• replication of plasmids with pMB1/colE1 replicons
  is not inhibited by cm
• Cm-treated cells stop growing but continue making
  these plasmids, this is a way to amplify plasmid
  copy numbers prior to a plasmid prep

62
Antibiotic resistance genes

• Tet A (C ) protein confers resistance to
  tetracycline (an inhibitor of protein synthesis)
  by pumping this antibiotic out of the cell
• Bacterial aminophosphotransferases confer
  resistant to kanamycins (aminoglycoside
  antibiotics that inhibit protein synthesis) by
  transferring the gamma phosphate of ATP to a 3
  hydroxyl group of the kanamycin

63
The ideal plasmid

• Confers a readily selectable phenotypic trait
• Has single sites for many restriction enzymes
• Low molecular weight
• -- Gives higher copy , stability, and
  transforming efficiency
• -- Can accept larger pieces of DNA
• -- Easier to handle (less susceptible to breakage)

64
pBR322

• The first widely useful cloning vehicle

Created using transposition and
restriction/ligation reactions
65
pBR322
Utility of pBR322 Clone into sites in the Tcr
gene, which allows identification of
recombinants--these will be amp resistant but tet
sensitive (initially plate on ampicillin, then
replica plate on tetracycline plates). But
pBR322 has low copy number, large size, and too
few options for cloning sites
66
Boldface indicates the restriction site is
present in only one site within the plasmid
67
pUC plasmidssecond generation cloning vectors

• Reduced size (about 2000 bp)
• Multiple cloning site (MCS, also called
  poly-linker) unique sites for lots of
  different restriction enzymes
• Very high copy number (mutation in RNA II)
• New blue-white screening tool for recombinants
  (alpha complementation is disrupted by foreign
  DNA in the MCS)

68
Alpha complementation
X-gal

• Plasmid encodes N-terminus of beta galactosidase
  (alpha fragment)
• Host strain encodes the C-terminus of beta
  galactosidase (omega fragment)
• Beta galactosidase function is only seen in the
  presence of both the N- and C-terminal fragments
• Beta gal function can be monitored by the
  cleavage of X-gal which yields a bright blue
  product (blue colonies on a plate)

Bright blue
69
An alpha complementing plasmid vector
(MCS)
pUC 19
DNA in the MCS interrupts the lacZ gene (no Beta
galactosidase)
70
Alpha complementation

• Plasmid encodes N-terminus of beta galactosidase
  (alpha fragment), with an MCS
• Foreign DNA in the MCS, no alpha fragment
• No alpha fragment, no B-gal
• No B-gal, no blue color (white colonies)

Colony without foreign DNA in MCS
pUC19 transformation plate
Colony with foreign DNA in MCS
71
Third generation cloning vectors specialized
plasmids

• Vectors containing bacteriophage RNA polymerase
  promoters for production of a specific RNA
  (probe synthesis, in vitro translation, etc.)
• Low copy number vectors for cloning of unstable
  or toxic genes
• Vectors designed for expression of specific
  proteins (for further purification and
  biochemical characterization). Proteins may be
  synthesized with tags to assist in purification

72
Transformation of E.coli with plasmid DNA

• E.coli strain must be antibiotic sensitive, best
  if it lacks restriction-modification systems
• Make cells take up DNA by
• Chemical competence
• Electroporation
• (natural competence--not E.coli though)

73
Chemically competent cells-basic method

• Grow cells to A600 of 0.4, spin to get cell
  pellet
• Resuspend cells in CaCl2 (100 mM), pellet again
• Resuspend in small volume of CaCl2/glycerol
• Freeze cells (-80C) or go straight to
  transformation protocol

74
Transformation of chemically competent cells
DNA binds to cells

• Mix DNA and competent cells, on ice for 30 min.
• Heat shock (42C) for 1.5 minutes
• Add growth media, 37C for 1 hour
• Plate on growth medium plus selection
  (antibiotic) for the plasmid
• Efficiency 106 - 107 cells/microgram plasmid DNA

DNA uptake by cells
Cells recover
Selection occurs
If cells are good
75
Ultra competent cells (chemical)

• 5 x 108 transformants/microgram plasmid
• See protocol 23 of Molecular Cloning ch. 1
• Treat with
• MnCl2
• CaCl2
• KCl
• Hexammine CoCl2
• Store in DMSO
• (protocol rather difficult, inconsistent)
• These can be bought

76
Transformation by electroporation

• gt 109 transformants/microgram DNA (ideally)
• Grow cells to A600 of 0.4
• Centrifuge and resuspend in water 10 glycerol
  (do this 4 times to reduce conductivity)
• Place cells with DNA in electrode-containing
  cuvette, deliver electrical pulse
• If there is arcing (sparks) transformation
  efficiency will be poor (uneven transfer of
  charge). To avoid this make sure the ion
  concentration is very low (less than 10 mM salt)

77
When cloning a piece of DNA consider 1) Choice
of vector what kind of plasmid vector to use
(which restriction sites can be used in the
vector)? 2) Ligating DNA to vector how will the
ligation reaction be set up to facilitate getting
what you want? 3) Moving DNA by transformation
what strain of E. coli will you transform into?
Which method for transformation? 4) Screening
for successful ligation products (recombinant
plasmid DNA) how will the recombinant plasmids
be identified?
78
Setting up a transformation--how will the
competent cells be treated?

1. No plasmid (negative control, nothing should grow
   on this plate)
2. Supercoiled plasmid of a known concentration (to
   determine efficiency of competent cells)
3. Vector DNA (dephosphorylated?) ligated without
   insert DNA (background transformants)
4. Vector DNA ligated with insert DNA (desired
   products)

79
Example outcome of a successful transformation
chemically competent cells

1. No DNA--No colonies
2. 2 nanograms (10-9 g, 10-3 micrograms) supercoiled
   plasmid DNA--500 colonies (efficiency of cells
   2.5 x 105 transformants per microgram DNA)
3. Vector alone--small number of colonies
4. Vector plus insert--larger number of colonies
   than for 3

80
Identifying recombinant plasmid-containing cells

• Alpha complementation most white colonies
  represent presence of insert DNA blocking
  functional beta galactosidase
• Increase in number of transformants in presence
  of insert vs. absence of insert
• Insert treated with alkaline phosphatase
• Directional cloning--preventing religation of
  vector
• Must screen colonies/plasmids for inserts,
  usually by PCR
• Confirm clones by sequencing

81

• Mobilizing DNA vectors for propagation in E.
  coli
• Plasmids
• Bacteriophage
• M13
• Lambda
• Cosmids and BACs