Researches About Mutagenic Potential Of D-/L-iso-T Bypass Catalyzed By DNA Polymerases

Nucleoside drugs derived from modified non-natural nucleosides have played an active role in antiviral and antitumor therapy.However,since the structures of the natural nucleosides are similar with the structures of the non-natural nucleosides,the selective relaxation of DNA polymerase may introduce modified nucleosides into the normal DNA strand.The selective relaxation of DNA polymerase may cause chemical damage to normal DNA in vivo.The incorporation of non-natural nucleotides and their derivatives in DNA strand may cause genetic variation.The incorporation may affect the biological function of normal nucleic acids.As a result,toxic side effects may occured.Our dissertation mainly studies the metabolism and side effects of a potential nucleoside drug molecule(isonucleoside)in vivo by incorporation of the isonucleoside into the DNA strand.The recognization ability of various polymerases to the potentially active isonucleosides has also been studied.The copy of the nucleic acids damaged by isonuclear under the action of polymerases has also been studied.The translession repaired or variation of the isonucleoside which placed in nucleic acid strand during normal cell replication process have also been studied.D-isonucleoside triphosphate can be recognized by the B-type DNA polymerase.DNA which was damaged by L-nucleoside has strong self-repairing ability in the presence of B-type DNA polymerase.Our research focused on the inhibition of human biological function by isonucleoside.The replication and transmission of DNA strand which was incorporated by isonucleosides were carefully studied.The specific toxic side effects of isonucleoside are revealed.1.The D-/L-iso-T phosphoramidite monomer was synthesized by the D-/L-isonucleoside-thymidine.The isonucleotide-modified nucleotide chain was synthesized by phosphoramidite method.2.The primer-extension of the D-/L-iso-T modified nucleotide was analyzed by polyacrylamide gel electrophoresis.Kinetic analysis has also been conducted.The results indicate that single or consecutive two D-iso-Ts site-modified nucleoside chains can be recognized by Taq and Vent(exo-)DNA polymerase.The L-iso-T site-modified nucleoside can not be recognized by the mentioned DNA polymerases.The rate of deoxyribonucleotide triphosphate(dNTP)incorporation decreases after incorporation of the D-iso-T.The results demonstrate that DNA polymerase has inhibitory effect on isonucleosides.3.The sequencing,fidelity analysis and thermodynamic stability of the D-iso-T site-modified DNA template were studied.The conclusions are(1)The thermodynamic stability of D-iso-T paired with A/G is higher than the other two base pairs.(2)Besides normal TA assignment,a sliding mutation in a single base has also been observed.The sliding mutation reveals a new inhibitory mechanism of the D-iso-T damage on the biological function of human.