chapter 5- dna modifying enzymes

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Enzymes that modify DNA are useful because they allow the investigator to manipulate DNA in defined ways - Polymerases elongate DNA molecules by adding free nucleotides to the 3’ ends (usually according to an opposite template strand) - Endonucleases cut DNA fragments in the middle of the molecule - Exonucleases degrade DNA from the ends - Ligases join loose ends of DNA together DNA modifying enzymes

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Page 1: Chapter 5- DNA Modifying Enzymes

Enzymes that modify DNA are useful because they

allow the investigator to manipulate DNA in defined

ways

- Polymerases elongate DNA molecules by adding

free nucleotides to the 3’ ends (usually according to

an opposite template strand)

- Endonucleases cut DNA fragments in the middle

of the molecule

- Exonucleases degrade DNA from the ends

- Ligases join loose ends of DNA together

DNA modifying enzymes

Page 2: Chapter 5- DNA Modifying Enzymes
Page 3: Chapter 5- DNA Modifying Enzymes

DNA polymerases exonuclease activities:

- Activity 3→5 exonuclease. ( proofreading activity)

allows the polymerase to correct errors by

removing a nucleotide that has been inserted

incorrectly.

- Activity 5→3 exonuclease activity is possessed by

some DNA polymerases.

DNA polymerases

Page 4: Chapter 5- DNA Modifying Enzymes

Proof reading activity

of the 3’ to 5’ exonuclease.

DNAPI stalls if the incorrect

ntd is added - it can’t add the

next ntd in the chain

Proof reading activity is slow

compared to polymerizing

activity, but the stalling of

DNAP I after insertion of an

incorrect base allows the

proofreading activity to

catch up with the polymerizing

activity and remove the

incorrect base.

Page 5: Chapter 5- DNA Modifying Enzymes

The types of DNA polymerases used in research:

DNA polymerase I: Unmodified E. coli enzyme .

Use: DNA labeling .

Klenow polymerase: Modified version of E.coli

DNA polymerase I

Use: DNA labeling

Page 6: Chapter 5- DNA Modifying Enzymes

The Enzymology

• In 1957, Arthur Kornberg demonstrated the

existence of a DNA polymerase - DNA

polymerase I

• DNA Polymerase I has THREE different

enzymatic activities in a single polypeptide:

• a 5’ to 3’ DNA polymerizing activity

• a 3’ to 5’ exonuclease activity

• a 5’ to 3’ exonuclease activity

of DNA Replication

Page 7: Chapter 5- DNA Modifying Enzymes

Functional domains in the Klenow Fragment (left) and DNA Polymerase I (right).

Page 8: Chapter 5- DNA Modifying Enzymes

DNA polymerase I

Page 9: Chapter 5- DNA Modifying Enzymes

Nick Translation

Page 10: Chapter 5- DNA Modifying Enzymes

Nucleases

Page 11: Chapter 5- DNA Modifying Enzymes

Exonucleases

Page 12: Chapter 5- DNA Modifying Enzymes

Figure 1. Prepare single-stranded template with Lambda Exonuclease.

Page 13: Chapter 5- DNA Modifying Enzymes

Figure. 1 Lambda

Exonuclease

selectively digests the

strand of a PCR

product produced

using a PCR primer

with a 5´-phosphate.

The resulting single-

stranded PCR product

can be used for SSCP

analysis or

sequencing.

Page 14: Chapter 5- DNA Modifying Enzymes

Nucleases

Endonucleases

Page 15: Chapter 5- DNA Modifying Enzymes

Endonucleases

I. Non specificII. Specific

e.g. S1 nuclease, from the fungus

Aspergillus oryzae

And Deoxyribonuclease I

(DNaseI), from Escherichia coli

e.g. Restriction endonucleases,

from many sources

Page 16: Chapter 5- DNA Modifying Enzymes

Endonucleases

I. Non specific

- S1 nuclease (Endonuclease specific for single-

stranded DNA and RNA, from the fungus Aspergillus

oryzae

� Use:Transcript mapping

- Deoxyribonuclease I (DNaseI) Endonuclease specific

for double stranded DNA and RNA, from Escherichia

coli

� Use:Nuclease footprinting

Page 17: Chapter 5- DNA Modifying Enzymes
Page 18: Chapter 5- DNA Modifying Enzymes

S1 nuclease protection

• digests only single-stranded RNA and DNA

Find introns:

genomic DNA

intron

Digest with S1

Run gel

exon 1 exon 2

antisense probe

exon 1

exon 2

Page 19: Chapter 5- DNA Modifying Enzymes

Endonucleases

II. Specific

e.g. Restriction

endonucleases: Sequence-

specific DNA endonucleases,

from many sources

� Use:Many applications

Page 20: Chapter 5- DNA Modifying Enzymes

Restriction Endonucleases

- Also called restriction enzymes

- Recognize, bind to, and cleave DNA molecules

at specific sequences (usually 4-6 base pairs in

length) but there are some that are 5, 8, or longer

- The double strand breaks can create ends that

are:

* Blunt, cutting both strands in the same place

* Sticky, with overhanging nucleotides on the 5’

or 3’ ends

Restriction endonuclease

Page 21: Chapter 5- DNA Modifying Enzymes

Restriction enzymes

• Over 10,000 bacteria species have been

screened for restriction enzymes

• Over 2,500 restriction enzymes have been found

• Over 250 distinct specificities

• Occasionally enzymes with novel DNA sequence

specificities are still found while most now prove

to be duplicates (isoschizomers) of already

discovered specificities.

Restriction endonuclease

Page 22: Chapter 5- DNA Modifying Enzymes

There are three types of restriction enzymes.

With Types I and III there is no strict control over the

position of the cut relative to the specific sequence in the

DNA molecule that is recognized by the enzyme. These

enzymes are therefore less useful .

Type II enzymes do not suffer from this disadvantage

because the cut is always at the same place, either within

the recognition sequence or very close to it

Restriction endonuclease

Page 23: Chapter 5- DNA Modifying Enzymes

Type II Restriction enzymes are endonucleases that

cut DNA at specific sites, and are most useful for

molecular biology research

Page 24: Chapter 5- DNA Modifying Enzymes

Restriction enzymes are

molecular scissors

Restriction enzymes

Page 25: Chapter 5- DNA Modifying Enzymes

• Restriction Enzymes scan the DNA code

• Find a very specific set of nucleotides

• Make a specific cut

Restriction enzymes

Page 26: Chapter 5- DNA Modifying Enzymes

Picking a palindromeWords that read the same forwards as backwards

Hannah

Level

Madam

hannaH

leveL

madaM

Restriction enzymes

Restriction enzymes recognize and make a cut within

specific palindromic sequences, known as restriction sites,

in the genetic code. This is usually a 4- or 6 base pair

sequence.

Page 27: Chapter 5- DNA Modifying Enzymes

Restriction Enzyme Recognition Sites

Restriction sites are general palindromic:

“Able was I, ere, I saw Elba”

Bam H1 site:5’-GGATCC-3’

3’-CCTAGG-5’

Restriction enzymes

Page 28: Chapter 5- DNA Modifying Enzymes

HaeIIIHaeIII is a restriction enzyme that searches the

DNA molecule until it finds this sequence of

four nitrogen bases.

5’ TGACGGGTTCGAGGCCAG 3’

3’ ACTGCCCAAGGTCCGGTC 5’

5’ TGACGGGTTCGAGGCCAG 3’

3’ ACTGCCCAAGGTCCGGTC 5’

Page 29: Chapter 5- DNA Modifying Enzymes

Once the recognition site was found

HaeIII could go to work cutting

(cleaving) the DNA

5’ TGACGGGTTCGAGGCCAG 3’

3’ ACTGCCCAAGGTCCGGTC 5’

Page 30: Chapter 5- DNA Modifying Enzymes

These cuts produce what scientists call

“blunt ends”

5’ TGACGGGTTCGAGG CCAG 3’

3’ ACTGCCCAAGGTCC GGTC 5’

Page 31: Chapter 5- DNA Modifying Enzymes

Restriction enzymes are named based on the bacteria in

which they are isolated in the following example for the

enzyme EcoRI:

E Escherichia (genus)

co coli (species)

R RY13 (strain)

I First identified Order ID'd in bacterium

Restriction enzymes

Page 32: Chapter 5- DNA Modifying Enzymes

Nomenclature of Restriction Enzymes

The 1st letter (in capital and italics) = first initial of

Genus name (from which the enzyme was isolated

• The 2nd and 3rd (in italics) = the first 2 letters of

the species name

e.g. Hin = Haemophilus influenzae

• The 4th letter (sometimes in italics) = strain or type

e.g. Hind = Haemophilus influenzae Rd

• The roman number followed is given to distinguish

different restriction and modification system in the

same strain

e.g. HindIII

Restriction enzymes

Page 33: Chapter 5- DNA Modifying Enzymes

5’ G AATTC 3’

3’ CTTAA G 5’EcoRI

EcoRII .....CCWGG

GGWCC.....

W=A or T

Page 34: Chapter 5- DNA Modifying Enzymes

“blunt ends” and “sticky ends”

Remember how HaeIII produced a “blunt end”?

EcoRI, for instance, 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’

Page 35: Chapter 5- DNA Modifying Enzymes

Eco RI Restriction Enzyme

Single stranded “nick”

Restriction enzymes

Page 36: Chapter 5- DNA Modifying Enzymes

Some more examples of restriction sites of

restriction enzymes with their cut sites:

HindIII: 5’ AAGCTT 3’

3’ TTCGAA 5’

BamHI: 5’ GGATCC 3’

3’ CCTAGG 5’

AluI: 5’ AGCT 3’

3’ TCGA 5’

Page 37: Chapter 5- DNA Modifying Enzymes

Restriction Enzyme Recognition Sites

BglII 5’ A-G-A-T-C-T

T-C-T-A-G-A 5’

Sau3A 5’ G-A-T-C

C-T-A-G 5’

BamHI 5’ G-G-A-T-C-C

C-C-T-A-G-G 5’

All these sticky ends

are compatible

Isoschizomers: In certain cases, two or more different enzymes may

recognize identical sites. (e.g. MboI also cleaves at GATC, and so is an

isochizomer of Sau3A.)

Page 38: Chapter 5- DNA Modifying Enzymes

Frequency of cutting of recognition enzymes

Sau 3A (GATC) cuts (¼)(¼)(¼)(¼) = once every 256 base

pairs (assuming G/C = A/T, which is often does not)

BamH1 (GGATCC) cuts (¼)(¼)(¼)(¼)(¼)(¼) = once every

~4Kb

HindII (GTPyPuAC) cuts (¼)(¼)(½)(½)(¼)(¼) = once

every ~1Kb

Restriction enzymes

http://tools.neb.com/NEBcutter2/index.php

Page 39: Chapter 5- DNA Modifying Enzymes

Human DNA cleaved with EcoRI Corn DNA cleaved with EcoRI

5’-C-G-G-T-A-C-T-A-G-OH

3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4

PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’

HO-G-T-C-G-A-T-G-C-5’

Ligation of compatible sticky ends

+

5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’

3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’

Complementary base pairing

+ DNA Ligase, + rATP

recombinant DNA molecule

5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’

3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’

Page 40: Chapter 5- DNA Modifying Enzymes

YIP M

EcoRI 5660

HindIII 1/ 6160

Eagl 542

Apal 2035

SmaI 2860PvuII 3547

PvuII 5116

SmaI 5’ ccc ggg 3’

Exercise1

How many base pairs in this plasmid?How mamy fragments will be produced if this plasmid is digested with PvuII?

Page 41: Chapter 5- DNA Modifying Enzymes

Agarose Gel Electrophoresis

_

+

DNA is negatively

charged from the

phosphate backbone

Visualize DNA with ethidium

bromide – fluoresces orange

ONLY when bound to DNA

Agarose mesh

Page 42: Chapter 5- DNA Modifying Enzymes

Gel Electrophoresis of DNA

Page 43: Chapter 5- DNA Modifying Enzymes

What is Gel Electrophoresis?

• Electro = flow of electricity, phoresis, from the Greek

= to carry across

• A gel is a colloid, a suspension of tiny particles in a

medium, occurring in a solid form, like gelatin

• Gel electrophoresis refers to the separation of charged

particles located in a gel when an electric current is

applied

• Charged particles can include DNA, amino acids,

peptides, etc

Page 44: Chapter 5- DNA Modifying Enzymes

Gel electrophoresis is a widely used technique for the

analysis of nucleic acids and proteins. Agarose gel

electrophoresis is routinely used for the preparation and

analysis of DNA.

Gel electrophoresis is a procedure that separates

molecules on the basis of their rate of movement through

a gel under the influence of an electrical field.

Gel electrophoresis

Page 45: Chapter 5- DNA Modifying Enzymes

Why do gel electrophoresis?

• When DNA is cut by restriction enzymes, the

result is a mix of pieces of DNA of different

lengths

• It is useful to be able to separate the pieces - i.e.

for recovering particular pieces of DNA, for

forensic work or for sequencing

Page 46: Chapter 5- DNA Modifying Enzymes

46

Gel with molecular weight marker

Page 47: Chapter 5- DNA Modifying Enzymes

Summary

• Restriction endonucleases recognize specific sequences in

DNA molecules and make cuts in both strands

• This allows very specific cutting of DNAs 4-7

• The cuts in the two strands are frequently staggered, so

restriction enzymes can create sticky ends that help to link

together 2 DNAs to form a recombinant DNA in vitro

Page 48: Chapter 5- DNA Modifying Enzymes

Plasmid vectors containing a polylinker

(a) Sequence of a polylinker that includes one copy of the recognition

site, indicated by brackets, for each of the 10 restriction enzymes

indicated. Polylinkers are chemically synthesized and then are inserted

into a plasmid vector. Only one strand is shown

Exercise

Page 49: Chapter 5- DNA Modifying Enzymes

1. The nucleotide sequence of a polylinker in a particular

plasmid vector is

GAATTCCCGGGGATCCTCTAGAGTCGACCTGCAGG

CATGC-

This polylinker contains restriction sites for BamHI , EcoRI ,

PstI , SalI , SmaI , SphI , and XbaI . Indicate the location of

each restriction site in this sequence.

2. A vector has a polylinker containing restriction sites in

the following order: HindIII , SacI , XhoI , BglII , XbaI , and

ClaI .

- Give a possible nucleotide sequence for the polylinker .

Page 50: Chapter 5- DNA Modifying Enzymes

C¯ CCGGGXmaI

T¯ CTAGAXbaI

GCATG¯ CSphI

CCC¯ GGGSmaI

G¯ TCGACSalI

GAGCT¯ CSacI

CTGCA¯ GPstI

G¯ AATTCEcoRI

G¯ GATCCBamHI

Recognition

SequenceEnzyme

Page 51: Chapter 5- DNA Modifying Enzymes

Nucleases

What is difference between DNase and RNase?

DNase cut DNARNases cut RNA

Page 52: Chapter 5- DNA Modifying Enzymes

RNases

Page 53: Chapter 5- DNA Modifying Enzymes

Ribonuclease H (RNase H)

Page 54: Chapter 5- DNA Modifying Enzymes

Replacement

Synthesis

Page 55: Chapter 5- DNA Modifying Enzymes

DNA fragments that have been generated by

treatment with a restriction endonuclease can be

joined back together again, or attached to a new

partner, by a DNA ligase. The reaction requires

energy, which is provided by adding either ATP or

NAD to the reaction mixture, depending on the

type of ligase that is being used.

DNA ligases

Page 56: Chapter 5- DNA Modifying Enzymes

DNA replication requires many

enzymes and protein factors

- Replisome

- Helicases

- Topoisomerases

- DNA-binding proteins

- Primases

- DNA ligases

Page 57: Chapter 5- DNA Modifying Enzymes

DNA ligases

Page 58: Chapter 5- DNA Modifying Enzymes

Application of DNA ligase

Page 59: Chapter 5- DNA Modifying Enzymes

Role of Phosphatase in DNA ligation

Page 60: Chapter 5- DNA Modifying Enzymes

Phosphotases & Kinases

Page 61: Chapter 5- DNA Modifying Enzymes

Flow of Genetic Information :

The Central Dogma of Molecular Biology

Alberts et al, 2002, p. 301

Reverse

transcriptase

DNA polymerase

Page 62: Chapter 5- DNA Modifying Enzymes
Page 63: Chapter 5- DNA Modifying Enzymes

- An enzyme that catalyses the synthesis of a

DNA strand from an RNA template.

- The produced DNA called complementry

DNA (cDNA)

* Present in retro virus & other RNA viruses

* Application: Used in RT-PCR (e.g.

detection of HCV-Ag)

Reverse transcriptase

Page 64: Chapter 5- DNA Modifying Enzymes

Reverse transcriptase