POLYMERASES

1. POLYMERASES M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.

2. Polymerases • DNA-dependent DNA polymerases Eg.Prokaryotic and Eukaryotic DNA polymerases • DNA-dependent RNA polymerases Eg. RNA polymerases and DNA primase • RNA-dependent DNA polymerases Eg. Reverse transcriptase • RNA-dependent RNA polymerases Eg. RNA replicase

3. RNA Replicase Reich et al. showed the RNA synthesis even in the presence of actinomycin-D (that inhibits the DNA-primed RNA synthesis) Baltimore and Franklin showed the viral production with specific cytoplasmic fractions Thus, RNA-primed RNA synthesis was demonstrated This enzyme has been named RNA-dependentRNA polymerase or RNA Replicase

4. All RNA viruses (except retroviruses) encode a protein since the hosts do not possess it • Structure of Qβ replicase: • Four subunits • 3 provided by E. coli host (Tu, Ts, and S1) • 1 in viral RNA genome (MW 65,000) • Function: • Synthesis in 5’ to 3’ direction • Lacksproofreading function • Requires RNA template • Specific for RNA of the virus

5. RNA Replicase was isolated by Spiegelman et al. from Qβ bacteriophage • This requires RNA template, Mg2+, rNTPs • Qβ replicase is tetramer consisting of one Rep molecule and three host proteins • Replicase copies viral (+) strand to generate a (-) strand

6. During synthesis, (-) strand is in contact with the (+) strand only at the polymerization site • Initiation of several (-) strand occurs before the first (-) is complete, and the replicative form is branched • The (-) strands are released and immediately used by the replicase to form (+) strands only

7. Reverse Transcriptase

8. Extension of the Central dogma

9. Temin and Baltimore (1970) independently demonstrated the presence of RT in several avian and mouse retroviruses • RT is a RNA-dependent DNA polymerase • It is used in rDNA technology to transcribe mRNA into dsDNA

10. RT has 3 enzymatic activities • It copies an RNA molecule to yield dsDNA-RNA, using a primer and joining dNTPs in a 3’-5’ linkage • It copies a primed ssDNA to form dsDNA • It degrades RNA in a DNA-RNA hybrid; this is called RNase H activity (3’ to 5’ exoribonuclease activity)

11. Structure and gene products

13. Retrovirus - HIV (1) Viral genome and reverse transcriptase enter cell.

14. Site of action of AZT and other reverse Transcriptase inhibitors (1) Viral genome and reverse transcriptase enter cell. (2) DNA copy synthesized by reverse transcriptase. RNA DNA

15. Site of action of AZT and other reverse Transcriptase inhibitors (1) Viral genome and reverse transcriptase enter cell. (2) DNA copy synthesized by reverse transcriptase. RNA DNA (3) RNA degraded; second DNA strand synthesized. DNA DNA

16. Site of action of AZT and other reverse Transcriptase inhibitors Host cell nucleus Host cell genome (4) DNA circularizes (unintegrated provirus) or integrase functions to incorporate DNA into host cell genome (integrated provirus). Site of action of antiretroviral drugs under development (1) Viral genome and reverse transcriptase enter cell. (2) DNA copy synthesized by reverse transcriptase. RNA DNA (3) RNA degraded; second DNA strand synthesized. DNA DNA

17. Site of action of AZT and other reverse Transcriptase inhibitors Host cell nucleus Host cell genome (4) DNA circularizes (unintegrated provirus) or integrase functions to incorporate DNA into host cell genome (integrated provirus). (5) With host cell activation, viral DNA is transcribed, yielding messenger RNAs and viral genome RNA. Site of action of antiretroviral drugs under development (1) Viral genome and reverse transcriptase enter cell. (2) DNA copy synthesized by reverse transcriptase. RNA DNA (3) RNA degraded; second DNA strand synthesized. DNA DNA

18. Site of action of AZT and other reverse Transcriptase inhibitors Host cell nucleus Host cell genome (6) Viral RNAs are translated, yielding viral enzymes (including protease) and structural proteins. (4) DNA circularizes (unintegrated provirus) or integrase functions to incorporate DNA into host cell genome (integrated provirus). (5) With host cell activation, viral DNA is transcribed, yielding messenger RNAs and viral genome RNA. Site of action of protease inhibitors Site of action of antiretroviral drugs under development (1) Viral genome and reverse transcriptase enter cell. (2) DNA copy synthesized by reverse transcriptase. RNA DNA (3) RNA degraded; second DNA strand synthesized. DNA DNA

19. Site of action of AZT and other reverse Transcriptase inhibitors Host cell nucleus Host cell genome (7) Viral membrane proteins are transported to host cell membrane. (6) Viral RNAs are translated, yielding viral enzymes (including protease) and structural proteins. (4) DNA circularizes (unintegrated provirus) or integrase functions to incorporate DNA into host cell genome (integrated provirus). (5) With host cell activation, viral DNA is transcribed, yielding messenger RNAs and viral genome RNA. Site of action of protease inhibitors Site of action of antiretroviral drugs under development (1) Viral genome and reverse transcriptase enter cell. (2) DNA copy synthesized by reverse transcriptase. RNA DNA (3) RNA degraded; second DNA strand synthesized. DNA DNA

20. Site of action of AZT and other reverse Transcriptase inhibitors Host cell nucleus Host cell genome (7) Viral membrane proteins are transported to host cell membrane. (6) Viral RNAs are translated, yielding viral enzymes (including protease) and structural proteins. (4) DNA circularizes (unintegrated provirus) or integrase functions to incorporate DNA into host cell genome (integrated provirus). (5) With host cell activation, viral DNA is transcribed, yielding messenger RNAs and viral genome RNA. Site of action of protease inhibitors Site of action of antiretroviral drugs under development (1) Viral genome and reverse transcriptase enter cell. (8) Final viral assembly and budding take place. (2) DNA copy synthesized by reverse transcriptase. RNA DNA (3) RNA degraded; second DNA strand synthesized. DNA DNA