28
KAI-OLAF NETZER
/soschizomeres
Asp718
Kpnl
GbTACC
CCATcr
GGTAch
CfATGG
Compatible ends
Mbol
BamHI
bATC
CTAGt
GbTACC
Blunt ends
5 ' - 3 ' 5 ' - 3 '
3 ' - 5 ' 3 ' - 5 '
Haelll
GG CC
CC GG
3' protruding
5'
3' 5'-3'
3'-5' 3'
5'
Pstl
CTGCA
G
G
ACGTC
3' recessed
5'-3' 5'
3'
3'
5' 3 ' - 5'
G
AATTC
EcoRI CTTAA
G
Fig. 3. Cleavage by lsoschizomeres
isadimer comprised of two identical31 kD subunits. Contact of the enzyme
with the major groove ofB-DNA (the classical double helix) leads to kinking
of the double helix in the center of the hexanucleotide target. The DNA is
unwound by 25 degrees, the major groove is widened 0.4 nm and the enzyme
enters the groove. A total of 12 hydrogen bonds is formed between the
alpha-helices of the enzyme and the two trinucleotides of GAA. The amino
ends of the alpha-helices interact with the negatively charged phosphate
backhone of the DNA and hydrolytic cleavage is initiated.