Effects of 2'-substituents of the first deoxyguanosine residue in the recognition sequence of EcoRI restriction endonuclease activity.
The effects of 2'-substituents of the first deoxyguanosine on EcoRI activity were examined using synthetic octadeoxynucleotides D(GG*AATTCC) containing 2'-substituted derivatives (G*), i.e., 2'-fluoro-2'-deoxyguanosine (dGfl), 2'-chloro-2'-deoxyguanosine (dGcl), and guanosine (rG). The overall structures of the octamers were very similar, as shown by CD and UV measurements, although their EcoRI reactivities were very different: 100% in 60 min for d(GGAATTCC) and d(GGflAATTCC), 5% in 24 h for d[G(rG)AATTCC], and no cleavage at all in 24 h for d(GGclAATTCC). However, the kinetics showed the octamers exhibit similar binding-affinity to the enzyme (10(-6)-10(-7) M). 31P-NMR analysis suggested the modified octamers change the phosphate backbone conformation in a duplex, since an unusual downfield-shifted signal in the spectra was commonly observed for the modified octamers at low temperature (i.e., a duplex state), which was shifted upfield at high temperature (i.e., a single strand state). The order of the differences was dGcl > rG > dGfl-containing octamers, coinciding with that of the vdW volume of 2'-substituents (Cl > OH > F) and the cleavage reactivities. These findings suggest that steric hindrance by the 2'-substituents causes of conformational change of the phosphate backbone close to the scissile bond, and then interferes with the EcoRI reaction.
Gohda
K
K
Faculty of Pharmaceutical Sciences, Osaka University.
Matsuo
N
N
Oda
Y
Y
Ikehara
M
M
Uesugi
S
S
D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003849:Deoxyguanosine / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D015246:Deoxyribonuclease EcoRI; D003849:Deoxyguanosine