Date of Award

Spring 5-5-2011

Degree Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Dr. Zhen Huang


DNA polymerase binds to the double stranded DNA and extends the primer strand by adding deoxyribonucletide to the 3’-end. Several reactions in the polymerase active site have been reported by Kornberg in addition to the polymerization. We observed DNA polymerase I can act as a pyrophosphatase and hydrolyze deoxyribonucletide. In performing the pyrophosphatase activity, DNA polymerase I requires to interact with RNA. RNA in general, was found to activate the DNA polymerase I as pyrophosphatase. This hydrolysis causes depletion of dNTP and inhibits DNA polymeration synthesis in vitro. In this RNA-dependent catalysis, DNA polymerase I catalyzes only dNTP but not rNTP. We have also observed that many other DNA polymerases have this type of the RNA-dependent pyrophosphatase activity. Our experimental data suggest that the exonuclease active sites most likely play the critical role in this RNA-dependent dNTP hydrolysis, which might have a broader impact on biological systems.

On the basis of the crystal structure of a ternary complex of RNase H (Bacillus halodurans), DNA, and RNA, we have introduced the selenium modification at the 6-position of guanine (G) by replacing the oxygen (SeG). The SeG has been incorporated into DNA (6 nt. - 6 nucleotides) by solid phase synthesis. The crystal structure and biochemical studies with the modified SeG-DNA indicate that the SeDNA can base-pair with the RNA substrate and serve as a template for the RNA hydrolysis. In the crystal structure, it has been observed that the selenium introduction causes shifting (or unwinding) of the G-C base pair by 0.3 Å. Furthermore, the Se-modification can significately enhance the phosphate backbone cleavage (over 1000 fold) of the RNA substrate, although the modifications are remotely located on the DNA bases. This enhancement in the catalytic step is probably attributed to the unwinding of the local duplex, which shifts scissile phosphate bond towards the enzyme active site. Our structural, kinetic and thermodynamic investigations suggest a novel mechanism of RNase H catalysis, which was revealed by the atom-specific selenium modification.

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