Enzymes

Enzymes are proteins biological catalysts help drive biochemical reactions

Enzyme names end with an ase (eg., endonuclease)

Restriction Endonucleases
Also called restriction enzymes 1962: molecular scissors discovered in in bacteria

E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA
3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially

Restriction Enzymes

Restriction Endonucleases

Restriction endonucleases RESTRICT viruses

Viral genome is destroyed upon entry

Restriction endonuclease = Restriction enzymes

Endo (inside), nuclease (cuts nucleic acid)

Restriction endonuclease recognizes a short and specific DNA sequence and cuts it from inside.
The specific DNA sequence is called recognition sequence

10/27/2012

Nomenclature

Smith and Nathans (1973) proposed enzyme naming scheme

three-letter acronym for each enzyme derived from

the source organism First letter from genus Next two letters represent species Additional letter or number represent the strain or serotypes

For example. the enzyme HindII was isolated from Haemophilus influenzae serotype d.

10/27/2012

Restriction Endonucleases
Named for bacterial genus, species, strain, and type

Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1

Few Restriction Enzymes

Target sequence Enzyme Organism from which derived (cut at *)

5' -->3'
Bam HI Eco RI Bacillus amyloliquefaciens Escherichia coli RY 13 G* G A T C C G* A A T T C

Hind III
Mbo I Pst I Sma I Taq I Xma I

Haemophilus inflenzae Rd
Moraxella bovis Providencia stuartii Serratia marcescens Thermophilus aquaticus Xanthamonas malvacearum

A* A G C T T
*G A T C CTGCA*G CCC*GGG T*CGA C*CCGGG

R-M System

Restriction-modification (R-M) system Endonuclease activity: cuts foreign DNA at the recognition site Methyltransferase activity: protects host DNA from cleavage by the restriction enzyme. Methylate one of the bases in each strand Restriction enzyme and its cognate modification system constitute the R-M system

10/27/2012

Uses for Restriction Enzymes

RFLP analysis (Restriction Fragment Length Polymorphism)

DNA sequencing
DNA storage libraries Transformation

Restriction Enzymes for Transformation

Human 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 Corn DNA cleaved with EcoRI

PO4-A-A-T-T-C-A-G-C-T-A-C-G-3 HO-G-T-C-G-A-T-G-C-5

Complementary base pairing

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

+ DNA Ligase, + rATP

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

recombinant DNA molecule

Protection of Self DNA

Bacteria protect their self DNA from restriction digestion by methylation of its recognition site. Methylation is adding a methyl group (CH3) to DNA. Restriction enzymes are classified based on recognition sequence and methylation pattern.

10/27/2012

Methylation

Classification
Evolved independently rather than diverging form a common ancestor Broadly classified into four Types

10/27/2012

Type I

Multi-subunit proteins Function as a single protein complex Contain

two R (restriction) subunits,
two M (methylation) subunits and one S (specificity) subunit

Cleave DNA at random length from recognition site

10/27/2012

Type III

Large enzymes Combination restriction-and-modification Cleave outside of their recognition sequences Require two recognition sequences in opposite orientations within the same DNA molecule No commercial use or availability

10/27/2012

Type IV

Cleave only modified DNA (methylated,

hydroxymethylated and glucosyl-hydroxymethylated

bases).

Recognition sequences have not been well defined Cleavage takes place ~30 bp away from one of the sites.

10/27/2012

Type II
Most useful for gene analysis and cloning

More than 3500 REs Recognize 4-8 bp sequences Need Mg 2+ as cofactor Cut in close proximity of the recognition site Homodimers ATP hydrolysis is not required

10/27/2012

Recognition Sequences

Each restriction enzyme always cuts at the same recognition sequence. Produce the same gel banding pattern (fingerprint) Many restriction sequences are palindromic. For example,
5 GAATTC 3 3 CTTAAG 5

(Read the same in the opposite direction (eg. madam, race car)

10/27/2012

blunt end

sticky end

Sticky End Cutters

Most restriction enzymes make staggered cuts Staggered cuts produce single stranded sticky-ends DNA from different sources can be spliced easily because of sticky-end overhangs.

HindIII

EcoRI

10/27/2012

Blunt End Cutters

Some restriction enzymes cut DNA at opposite base They leave blunt ended DNA fragments These are called blunt end cutters

AluI

HaeIII

10/27/2012

Restriction Enzyme Use

Discovery of enzymes that cut and paste DNA make genetic engineering possible. Restriction enzyme cuts DNA and generates fragments Ligase joins different DNA fragments

DNA fragments from different species can be ligated (joined) to create Recombinant DNA

10/27/2012

How does it Look after Restriction Digestion

Genomic DNA Digest Plasmid DNA Digest

10/27/2012

Restriction Maps

Useful but now most commonly generated by computer from actual DNA sequence

Restriction Fragment Length Polymorphism (RFLP) Analysis

Suspect

Evidence

DNA Fingerprintingused in modern forensics

Victim

2. DNA Modifying Enzymes a DNA Polymerase I

5-3 Exonuclease Activity 3-5 Exonuclease Activity DNA Polymerase Activity

The Klenow Fragment

Klenow fragment used for DNA sequencing

Reverse Transcriptase (RTase)

b nucleases

Deletion analysis using Bal 31 nuclease

c enzymes that modify the ends of DNA molecules i Terminal transferase

ii Polynucleotide kinase

iii Alkaline Phosphatase: BAP and CIP

3 DNA ligase

Mechanism of DNA ligase

DNA ligase used for both blunt and cohesive end ligation