Restriction Enzymes

RESTRICTION ENZYMES

Cell and Molecular Biology Lab Department of Biological Sciences UST College of Science Post Lab Discussion # 5

Restriction Enzymes  Restriction Endonucleases  Cleave DNA at specific sequences  Responsible for host-controlled restriction modification or phenotype modification (in bacteria)

 growth of phages are “restricted” to certain strains (Luria, 1950)  “Restriction” – bacterial ‘immunity’ against nonself, unmethylated DNA  Cornerstone of molecular biology  1978 Nobel Prize in Medicine to Werner Arber, Daniel Nathans, Hamilton Smith

BACTERIAL RESTRICTION ENZYMES

 With two components:  Restriction endonuclease - sequence specific nucleases  DNA methylase – modifies DNA by methylation (prevents cleavage of own DNA)  > 900 known REs  Isolated from 230 species of bacteria

RE PROPERTIES Property

Type I

Type II

Type III

Restriction and Modification

Single multifunctional enzyme

Separate nuclease and methylase

Separate enzymes sharing common subunit

Nuclease subunit structure

Heterotrimer

Homodimer

Heterodimer

Cofactors

ATP, Mg2+, SAM

Mg2+

Mg2+ (SAM)

DNA cleavage requirement

Two recognition sites in any arientation

Single recognition site (palindrome)

Two recognition sites in a head to head orientation

Site of methylation At recognition site

At recognition site

At recognition site

DNA translocation Yes

No

No

NOMENCLATURE  First letter (capital letter) – first letter of the genus where RE was isolated  Second and third letter (small letters) – first two letters of the species name (specific epithet) where RE was isolated  Fourth letter – first/second letter of strain name of organism where RE was isolated  Roman numeral – number (according to order of discovery) of RE isolated from the species.  Examples:  EcoR I – Escherichia coli (RY13 strain)  Hind III – Haemophilus influenzae (Rd strain)  Sma I – Serratia marcescens  Taq I – Thermophilus aquaticus  Kpn I – Klebsiella pneumoniae

RECOGNITION SITES  Sequence specific  Variable length  Recognize mostly palindromic sequences (4-8 bp)  EcoR I - GAATTC CTTAAG  May be interrupted by 1-9 nucleotides with no base specificity  Sfi I - GGCCNNNNNGGCC  Some allow ambiguity in the palindrome  Acc I – GTMKAC (where M = A or C and K = G or T)  Some (Type IIs) do not require palindromic sequences  Mbo II - 5’….GAAGA……3’

RE DIGESTION ACTIVITY  

Affedted by pH, temperature, salts and ION concentration An Enzymatic Unit (u)- the amount of enzyme required to digest 1 ug of DNA under optimal conditions: 3-5 u/ug of genomic DNA ;1 u/ug of plasmid DNA (Stocks typically at 10 u/ul)

ISOSCHIZOMERS BglII

5’ A-G-A-T-C-T T-C-T-A-G-A 5’

Sau3A

BamHI

5’ G-A-T-C C-T-A-G 5’

5’ G-G-A-T-C-C C-C-T-A-G-G 5’

In certain cases, two or more different enzymes may recognize identical sites All these sticky ends are compatible

“STAR” ACTIVITY  Altered or relaxed specificity  cleaving sequences similar (not identical) to their defined recognition sequence  RE’s with known star activity:  Apo I , Ase I , BamH I, BssH II, EcoR I, EcoR V, Hind III, Hinf I, Pst I, Pvu II, Sal I, Sca I, Taq I, Xmn I

 Caused by:  High units to µg of DNA ratio [Varies with each enzyme, usually >100 units/µg]  Low ionic strength [<25 mM]  High pH [>pH 8.0]  Presence of organic solvents [DMSO, ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane]  Substitution of Mg++ with other divalent cations [Mn++, Cu++, Co++, Zn++]

DIGESTION WITH MULTIPLE REs  If with compatible buffers: Digest with both enzymes in the same buffer.  If with slightly incompatible buffers: Cut with one enzyme, then alter the buffer composition and cut with the second enzyme.  If with totally incompatible buffers: Perform one digestion, recover the DNA (usually by precipitation) and resuspend in the buffer appropriate for the second enzyme.

RESULTS

4 bio 2

4 bio 3

4 bio 4

4 bio 5

4 bio 6

5000 bp

4969bp

3000

1000 750 500 250

Clone 2B

Clone 2A

Clone 1B

Clone 1A

5000bp marker

Clone 2B

Clone 2A

Clone 1B

Clone 1A

5000bp marker

Expression of Recombinant Blo t 11-fD in Escherichia coli

5000 bp 3000

666bp

1000 750 500 250

666bp

PCR products RE digested Plasmids using BamHI and XhoI

Verification of fD gene insert Plasmid pGEX-4T-1