1. 1. DNA REPLICATIONDNA REPLICATION Guided By- Dr.Cletus DSouza Dept. Of Biochemistry Presented By- Narayan Prahlad V VIth Semester Dept.of Molecular Biology
2. 2. ContentsContents Introduction Chemistry of DNA Synthesis Extension of 3-OH of Primer Enzymes involved in replication Mechanism of Replication Proof Reading Conclusion Reference Acknowledgement
3. 3. INTRODUCTIONINTRODUCTION
4. 4. Chemistry of DNA SynthesisChemistry of DNA Synthesis Requires 4 Deoxynucleoside Triphosphates (dGTP,dCTP,dATP,dTTP). Have three Phosphate groups-attached via 5-OH & 2 deoxyribose. Second essential substrate- Primer:Template Junction Primer- Should have exposed 3-OH.
5. 5. Extension of 3-OH of PrimerExtension of 3-OH of Primer New chain grows-extension of 3 end of primer-Phosphodiester bond is formed by SN2 mechanism.
6. 6. EnzymesEnzymes DNA Polymerase- Mainly catalyses DNA synthesis. Multimolecular forms of this enzyme- DNA Pol-I, DNA Pol-II, DNA Pol-III DNA Pol-I: Polymerase Activity. 5 to 3 Exonuclease Activity-Used to remove primer and replace with DNA. DNA Pol-III: Core replication enzyme. Holoenzyme Has proof reading capabilities(3 to 5 exonuclease activity).
7. 7. Polymerase Polymerase clamp loader Sliding clamp 3'-5' exonuclease
8. 8. DNA Pol-III The fingers and the thumb are composed-helices. The incoming dNTP is in red (for the base and the deoxyribose) and yellow (for the triphosphate moiety). The template strand of the DNA is shown in dark gray, and the primer strand is shown in light gray.
9. 9. Continued..Continued.. Helicase- Hexameric assembly of 6 identical subunits. Uses ATP (Hydrolysis)-unwind DNA. Sliding Clamp- Part of DNA Pol-III (-Subunit). Encircles and slides along DNA-tethering it to template.
10. 10. Continued..Continued.. Single Strand DNA Binding Protein (SSB)- Tertamer of identical subunits. Binds to ssDNA-preventing it from recoiling. DNA Primase- Synthesis of Oligoribonucleotide(Primer) -extended by DNA Pol-III. Ligase- Seals the gaps between Okazaki fragments-forming a continuous strand.
11. 11. Mechanism of ReplicationMechanism of Replication STEP-1 - UnwindingUnwinding of DNA by Helicaseof DNA by Helicase
12. 12. Step-2- Binding of SSBs to ssDNABinding of SSBs to ssDNA
13. 13. Step-3 Binding of TopoisomeraseBinding of Topoisomerase
14. 14. Step-4 Action of DNA Pol-IIIAction of DNA Pol-III
15. 15. Sliding ClampSliding Clamp
16. 16. Thrombone ModelThrombone Model In order to explain synthesis on lagging strand- Thrombone model was proposed.
17. 17. Synthesis of DNA on Lagging StrandSynthesis of DNA on Lagging Strand
18. 18. Action of DNA Pol-I & LigaseAction of DNA Pol-I & Ligase DNA Pol-I replaces RNA fragments with DNA and fills the gaps. Ligase joins the fragments Phosphodiester bonds and forms a continuous strand.
19. 19. Proof ReadingProof Reading DNA Pol-3. When a mismatched nucleotide comes-rate of addition of nucleotide by polymerase slows down. Exonuclease catalytic site in DNA Pol-3. Error rate- 1 in 1010 bp added, occasionally- 1 in 105 .
20. 20. ConclusionConclusion DNA replication is a very important phase in cell cycle. Replication is a must so as to ensure the equal distribution of genetic material to daughter cells. Thus all enzymes have to coordinate well in order for replication to occur successfully.
21. 21. ReferencesReferences Molecular Biology of Gene by Watson;7th edition; Cold Spring Harbour Laboratory Press, Cold Spring Harbour New York. Biochemistry 5th edition by Lubert Stryer;W.H Freeman Company. Molecular Cell Biology by Lodish;6th edition;W.H Freeman and Company;New York. http://sites.fas.harvard.edu/~biotext/animations/replication1.swf
22. 22. AcknowledgementAcknowledgement I would like thank my guide Dr.Cletus DSouza for his valuable guidance. I would also like to thank our course coordinator Dr.N.S Devaki for providing me this opportunity to present this seminar. Thank You all for your patient listening.