Restriction Enzymes Lecture 15: 1 11/20/ Definition: enzymes that recognize specific double-stranded sequences and hydrolyze the phosphodiester bonds on both strands. 2. How many REs have been known? ~ How is the activity of a RE defined? The mount of the enzyme required to digest one microgram of lamda DNA in one hour at the optimum conditions for the enzyme. 4. How many types of RES? 3. Type I and III REs have both endonuclease and methylase activities, cutting a random sequence away from the recognition sequence. Type II REs carry endonuclease activities. They cleave a specific sequence site within the recognition site. 5. Palindromes are common in the recognition sites of REs. EcoRI 5’-GAATTC-3’ 3’-CTTAAG-5’ 6. The rules for naming REs EcoRI is the first (I)restriction enzyme isoltated from Escherichia coli RY 13 strain.

7. Frequencies of Restriction Sites in a given sequence 4 nucleotides: G, A, T, C. If the recognition sequence is 1 nucleotide long, then a sequence of 4 nucleotides should contain one site of the RE. 4 1 =4. G, A, T, and C. How about 2: 4 2 =16 G A T C GGG GA GT GC A AG AA TT AC T TG TA TT TC C CC CA CT CC Let’s try 3: 4 3 =64 (triplet codons) GG GA GT GC AG AA TT AC G GGG GGA GGT GGC GAG GAA GTT GAC A AGG AGA AGT AGC AGA AAA ATT AAC T TGG TGA TGT TGC TAG TAA TTT TAC C CGG CGA CGT CGC CAG CAA CTT CAC TG TA TT TC CC CA CT CC G GTG GTA GTT GTC GCC GCA GCT GCC A ATG ATA ATT ATC ACC ACA ACT ACC T TTG TTA TTT TTC TCC TCA TCT TCC C CTG CTA CTT CTC CCC CCA CCT CCC Restriction Enzymes Lecture 15: 2 11/20/2006

Let’s try 3: 4 3 =64 (triplet codons) GG GA GT GC AG AA TT AC G GGG GGA GGT GGC GAG GAA GTT GAC 1 A AGG AGA AGT AGC AGA AAA ATT AAC 2 T TGG TGA TGT TGC TAG TAA TTT TAC 3 C CGG CGA CGT CGC CAG CAA CTT CAC 4 TG TA TT TC CC CA CT CC G GTG GTA GTT GTC GCC GCA GCT GCC 5 A ATG ATA ATT ATC ACC ACA ACT ACC 6 T TTG TTA TTT TTC TCC TCA TCT TCC 7 C CTG CTA CTT CTC CCC CCA CCT CCC 8 For the 4-base cutters: 4 4 =256 (=32X8=256) The smallest sequence recognized by any REs is at least 4 nucleotides long. GGG GGA GGT GGC GAG GAA GTT GAC 1 G GGGG GGGA GGGT GGGC GGTG GGAA GGTT GGAC A AGGG AGGA AGGT AGGC AGAG AGAA AGTT AGAC T TGGG TGGA TGGT TGGC TGAG TGAA TGTT TGAC C CGGG CGGA CGGT CGGC GAG CGAA CGTT CGAC ………, For a sequence of 256 nts, there is going to be at least one site for a 4 base recognition RE. Restriction Enzymes Lecture 15: 3 11/20/2006

Restriction Enzymes Lecture 15: 4 11/20/2006 Useful 4 cutters: 1. Sau3A: 5’…..GATC…...3’ 3’…..CTAG…...5’ Isolated from Staphylococcus aureus 3A 2. MboI MboI: 5’…..GATC…...3’ 3’….CTAG…...5’ Isolated from Moraxella bovis Isoschizomers: REs that recognize the same sequence are isochizomers. Sau3A and MboI are used to produce a partial digestion of a complex DNA in creating genomic DNA libraries. 3. Dpn I 5’…G m A TC…3 3’…C T m AG…3 Dpn I is often used in site-direted mutagenesis. For 6-cutter REs, 4 6 = 4096 nts These REs are frequently used to clone the genes into plasmids. For 8-cutter RE, 4 8 =65,536 nts Used at the flanking sites of cloning. Not I: 5’…..GCGGCCGC……3’ 3’…..CGCCGGCG……5’

Restriction Enzymes Lecture 15: 5 11/20/2006 For 8-cutter RE, 4 8 =65,536 nts Used at the flanking sites of cloning. Not I: 5’…..GCGGCCGC……3’ 3’…..CGCCGGCG……5’ From Nocardia otitidis-cavarun Asc I: 5’….GGCGCGCC……3’ 3’…..CCGCGCGG……5’ 8. Three types of ends are produced by RE digest: 1)Blunt ends: 5’-CCCGGG-3’ 3’-GGGCCC-5’ Sma I, Serratia marcescens 5’-GATATC-3’ 3’-CTATAG-5’ EcoR V, Escherichia coli 2) Sticky ends (5’-protruding or overhang) 5’-GGATCC-3’ 3’-CCTAGG-5’ BamH I, Bacillus amyolquefaciens H 3) Sticky ends (3’-protruding or overhang) 5’-GGTACC-3’ 3’-CCTTGG-5’ Kpn I: Klebsiella pneumoniae

Restriction Enzymes Lecture 15: 6 11/20/ Sticky ends can be converted into blunt ends or partially filled in: 5’-GGATCC-3’ 3’-CCTAGG-5’ BamH I 5’-G-3’ Fill-in buffer w/dGTP, dATP, dTTP, and dCTP, Klenow 3’-CCTAG-5’ 5’-GGATC-3’ 3’-CCTAG-5 5’-AAGCTT-3’ 3’-TTCGAA-5’ Hind III 5’-A-3’ Fill-in buffer w/dGTP, dATP, and [α- 32 P]dCTP, Klenow 3’-TTCGA-5’ 5’-AAG*C-3’ 3’-TTCGA-5’

Restriction Enzymes Lecture 15: 7 11/20/2006 T7 DNA Polymerase can convert 5’-overahng or 3’ overhang into blunt ends due to its DNA-dependent DNA polymerase and 3’- exonuclease (stronger than that of Klenow). 5’-GGTACC-3’ Kpn I 5’-GGTAC-3’ 3’-CCTTGG-5’ 3’-C-5’ 5’-GGTAC-3’ T7 DNA polymerase 5’-G-3’ 3’-C-5’ 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.

10. Activities of some restriction enzymes are methylation- dependent. CH3 5’……GATC……...3’ 5’……CTAG……...3’ CH3 This property is exploited to develop a site-directed mutagenesis (Quickchange Site-Directed Mutagenesis kit from Stratagene). (See transparency) 11. RFLP and RAPD Restriction Fragment Length Polymorphism (RFLP) Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another. Restriction Enzymes Lecture 15: 8 11/20/2006 cystic fibrosis (CF)