Restriction Endonucleases David Peterson

Structure of DNA and RNA

DNA RNA Protein Information Flow in Cells

DNA is a Double Helix

Phage 48,512 bp dsDNA virus that grows on E. coli. Life cycle ~ 30 min at 37°C typical burst ~ particles

Bacteriophage infection: 1 → 300 → 10,000 → 3,000,000 → 10 9 ! 30’ 30’ 30’ 30’ Bacterium Phage DNA Typical phage infection cycle: 1. Phage DNA penetrates bacterial envelope 2. Bacterial DNA is disrupted & phage DNA is replicated 3. Phage structural proteins are synthesized 4. Heads, tails, fibers made and assembled into particles 5. Cell lysis, release of ~ 100 – 300 phage particles 30 min

1000’s of phage particles attacking an E. coli cell

Bacteriophages Make Plaques

Restriction Endonucleases Type 1 Cuts at random position relative to recognition sequence (sometimes far away) Type II Cuts at recognition sequence Type III Cuts at fixed distance from recognition sequence

SV40 DNA Cut with Hin cII

Palindromes Madam, I’m Adam. Gary knits a stinky rag. Harass sensuousness, Sarah. Go hang a salami, I’m a lasagna hog.

Most Restriction Enzymes Recognize Palindromes GATATC ||||||||||||||||||| CTATAG 5’ 3’

EcoRV Bound to DNA 1AZ0

Methylation sites

Most Restriction Enzymes Recognize Palindromes GATATC ||||||||||||||||||| CTATAG 5’ 3’

EcoRV Bound to DNA 1AZ0

SV40 DNA Cut with Hin cII

Restriction Enzymes Aid in DNA Cloning

Dan Nathans and Hamilton Smith Werner Arber Nobel Prize in Physiology or Medicine 1978