110 Pingoud, Alves, and Geigersame DNA sequence. They all cleave the DNA within or next to the
recognition site producing "sticky" (with a 5' or 3' overhang) or"blunt"
ends, viz.
-GpTpCpGpApC-
SalI
-CpApGpCpTpG--G pTpCpGpApC-
+
-CpApGpCpTp G-
(stickyend with5'overhang)-CpGpApTpCpG-
PvuI
-GpCpTpApGpC--CpGpApT pCpG-
+
-GpCp TpApGpC-
(stickyend with3'overhang)-GpApTpApTpC- -GpApT pApTpC-
EcoRV ~ +
-CpTpApTpApG- -CpTpAp TpApG-
(blunt end with no overhang) (2)
Altogether, about 130 different specificities have been described.
Different strains of individual species often contain different restric-
tion enzymes: in E. coli, e.g., 141 restriction enzymes with 40 different
specificities are known (24). Few restriction enzymes have been shown
to be part of a restriction/modification system. Even fewer have been
purified to homogeneity and characterized biochemically. Most of our
knowledge on the mechanism of action of restriction endonucleases
rests on studies of these enzymes.
2.1. Purification
Bacteria that produce Type II restriction enzymes usually synthe-
size very small amounts of these proteins. Typically, between 1 and 10
mg of the homogeneous enzyme can be isolated/kg wet cell paste in a
multistep purification scheme. The isolation procedure in general involves
breaking up the cells by enzymatic or mechanical procedures, high-
speed centrifugation, removal of nucleic acids by precipitation with
polyethyleneimine or binding to DEAE-cellulose at an ionic strength
that prevents binding of the restriction enzyme to the DNA, and a
series of chromatographic steps. Phosphocellulose, hydroxyapatite,
and heparin-agarose chromatography seem to be particularly suited,
and have been widely employed, for the purification of restriction