Restriction Enzymes

1
Restriction Enzymes

• Tam Han
• Nwamaka Okagbue

2
Intro into DNA Enzymology

• History
• 1955 Arthur Kornberg and colleagues isolated DNA
  polymerase
• 1966 B. Weiss and C.C. Richardson isolated DNA
  ligase
• 1968 H.O. Smith, K.W. Wilcox, and T.J. Kelley
  isolated and characterized the first sequence
  specific restriction enzyme
• Phage growth restriction
• Phages grow and infect one strain, but not the
  other strain
• Methylation
• Not modified get degraded by hosts enzymes
• Methylase and Nuclease
• Late 1960s, Stewart Linn and Werner Arber
  isolated two types of enzymes responsible for
  phage growth restriction in E. coli bacteria.
• Methylase cleaved methylated DNA
• Restriction nuclease cleaved unmethylated DNA
  at a wide variety of sites
• Site-specific nuclease
• 1968, Johns Hopkins University, H.O. Smith, K.W.
  Wilcox, and T.J. Kelley first restriction
  nuclease whose functioning depended on a specific
  DNA nucleotide sequence
• HindII from Haemophilus influenzae bacteria
• Always cut at a particular point within a
  specific sequence of six base pairs
• 5 GTAC 3
• 3 CATG 5

3
What are they?

• Restriction enzymes are DNA-cutting enzymes found
  in bacteria (and harvested from them for use).
• Restriction endonucleases
• A restriction enzyme recognizes and cuts DNA only
  at a particular sequence of nucleotides.
• Bacteria prevent their own DNA from being cut by
  modifying their nucleotides via DNA methylation.

4
How REs cut DNA?

• Blunt ends
• Cleave opposing phosphodiester bonds no unpaired
  bases on ends
• Sticky ends
• Staggerd cuts of two DNA strands leaving unpaired
  nucleotides at the ends
• Base-pair with complementary sticky ends

5

• Three Types of Restriction Enzymes
• Type I cleave DNA at random sites that can be
  more than 1,000 bp from the recognition sequence.
• Type II first isolated in 1970 (Hamilton Smith),
  require no ATP and cleave DNA within the
  recognition sequence.
• Type III cleave about 25 bp from the recognition
  sequence.
• Nomenclature

6

• Restriction mapping
• The process of obtaining structural information
  on a piece of DNA by the use of restriction
  enzymes.
• A restriction map is a description of restriction
  endonuclease cleavage sites within a piece of
  DNA.
• Restriction mapping involves digesting DNA with a
  series of restriction enzymes and then separating
  the DNA fragments by agarose gel electrophoresis.
  
• The distance between enzyme sites can be
  determined by the size of the DNA fragments.

7
Restriction sites

• Palindromes sequences can be read the same way
  in either direction

8
Sources

• Nelson, David L., Michael M. Cox. Lehninger
  Principles of Biochemistry. 4th Ed. DNA-Based
  Information Technologies. pp.308-09.
• Peters, Pamela, Ph.D., Access Excellence,
  Genentech, Inc. lthttp//www.accessexcellence.org/A
  E/AEC/CC/restriction.phpgt
• Roberts, RJ, Belfort M, Bestor T, Bhagwat AS,
  Bickle TA, Bitinaite J, Blumenthal RM, Degtyarev
  SKh, Dryden DT, Dybvig K, Firman K, Gromova ES,
  Gumport RI, Halford SE, Hattman S, Heitman J,
  Hornby DP, Janulaitis A, Jeltsch A, Josephsen J,
  Kiss A, Klaenhammer TR, Kobayashi I, Kong H,
  Krüger DH, Lacks S, Marinus MG, Miyahara M,
  Morgan RD, Murray NE, Nagaraja V, Piekarowicz A,
  Pingoud A, Raleigh E, Rao DN, Reich N, Repin VE,
  Selker EU, Shaw PC, Stein DC, Stoddard BL,
  Szybalski W, Trautner TA, Van Etten JL, Vitor JM,
  Wilson GG, Xu SY (April 2003). "A nomenclature
  for restriction enzymes, DNA methyltransferases,
  homing endonucleases and their genes". Nucleic
  Acids Res. 31 (7) 180512. doi10.1093/nar/gkg274
  . PMID 12654995. lthttp//en.wikipedia.org/wiki/Res
  triction_enzymegt
• Smith, H.O., D. Nathans (December 1973). "Letter
  A suggested nomenclature for bacterial host
  modification and restriction systems and their
  enzymes". J. Mol. Biol. 81 (3) 41923. PMID
  4588280. lthttp//en.wikipedia.org/wiki/Restriction
  _enzymegt
• Dr. Jordan. Molecular Biology Tools. Lecture 1C.
  Slide 7.