Restriction/Modification

  1. The phenomenon with lambda and different E. coli strains
    1. efficiency of plating
    2. Luria's results with lambda

      3. Bacterial strain

       

      E.O.P.

       

       

      lambda(K-12)

      lambda(B)

      lambda(C)

      K12

      1

      10-4

      10-4

      B

      10-4

      1

      10-4

      C

      1

      1

      1

  2. Restriction/modification with K12 and B strains
    1. restriction is the phenomenon that leads to the low E.O.P.
      1. incoming viral DNA is "restricted" in the unfamiliar host strain
      2. like a type of immunity to viral infections
    2. modification is what happens to the few phage that escape restriction
      1. in other words, it is the reason that the E.O.P. is 10-4 and not zero
      2. the phage has been "modified" to survive in the new host strain
      3. this is not a mutation (e.g., an extended host range mutation), because the EOP keeps flip-flopping back and forth with every change of host strain.
  3. Molecular basis of restriction/modification
    1. had to wait until the technology was available
      1. Arber 1965-68
        1. showed that foreign DNA is degraded to small pieces in the restriction processs
          1. an enzymatic process of the host
          2. hence the term "restriction endonuclease", which we use all the time today
        2. the modification that protects the foreign DNA is methylation
        3. Arber went on the characterize the hsd (host specificity determinant) of E. coli
      2. hsd is an example of a Type I restriction endonuclease
        1. consists of three subunit types with division of labor
          1. HsdR is the restriction enzyme, an endonuclease
          2. HsdM is the methylation subunit
          3. HsdS is the site recognition subunit (and the site is a specific sequence in the DNA)
            1. for EcoB1 it's TGA(N8)TGCT, with the A closest to the middle picking up the methyl group
            2. for comparision,  the EcoK1 site is5'  AAC(N6)GTGC
        2. the way is should work is that the state of incoming foreign DNA determines its fate
          1. fully methylated DNA is not cut by HsdR
          2. hemimethylated DNA is methylated, and not cut
          3. unmethylated DNA is chopped up
            1. actual site of endonuclease cleavage is far from the recognition site
            2. what's worse, it is not always the same site each time (that is, HsdR seems to choose at random where it cuts)
    2. Unlike those that came after him, Arber was not able to capitalize on restriction endonuclease properties
      1. Daniel Nathans and Hamilton Smith 1970
        1. colleagues at Johns Hopkins
          1. Ham Smith isolated HindII, the first characterization of a Type II restriction endonuclease
          2. Nathans was at the same time perfecting gel electrophoresis of DNA fragments
          3. They determined the sequence of the HindII recognition (and cut, and modification) site
              4. 5' GTPyPuAC 3'
              3' CAPuPyTG 5'
            1. enzyme cut right down the middle (at the two fold symmetry axis, notice?)
            2. methylation of "A"s preventing cutting
        2. more on Type II restriction endonucleases
          1. single subunit has the site recognition and cutting, and another subunit does the methylating
          2. subunits can work independently
            1. this is, to quote Arthur Kornberg "one of nature's greatest gifts to biochemists"
            2. it's actually gets even better, since many Type II enzymes leave cohesive ends.  More on that later.
          3. properties and notation, Type II
            1. named after organism
            2. 4 base, 6 base, 8 base cutters
            3. compatible enzymes have different recognition sites but leave the same ssDNA overhang
              1. EcoRI and ApoI
              2. BamHI and Sau3AI
            4. isoschizomers
        1. biological role of type IIs - ?
        2. Type III restriction endonucleases are often found on viral genomes, eg., EcoPI
          1. more like type I than type II in cofactor requirements
          2. not useful
        3. Nathans used HindII to generate the first physical map (restriction map) of DNA
          1. SV40 story, and a Nobel Prize, richly deserved, for Arber, Nathans, and Smith
          2. restriction maps and how to make them
      1. Herb Boyer, EcoRI, and sticky ends
        1. EcoRI is a six base cutter isolated from E. coli, of course
        2. sequence and cut site is
          3.              5' G'AATTC
                       3' CTTAA'G
          1. notice that the single stranded overhangs are complementary
          2. this is true for all EcoRI sites, no matter the source of DNA
      2. Boyer, Cohen and Berg and the beginnings of recombinant DNA technology