Effects of 2'-Substituents of the First Deoxyguanosine Residue in the Recognition Sequence on EcoRI Restriction Endonuclease Activity

抄録

The effects of 2'-substituents of the first deoxyguanosine on EcoRI activity were examined using synthetic octadeoxynucleotides d(GG*AATTCC) containing 2'-substituted deriva-tives (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 the steric hindrance by the 2'-substituents causes a conformational change of the phosphate backbone close to the scissile bond, and then interferes with the EcoRI reaction.