DNA metabolism
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This presentation is meant for MBBS, MD and BDS students. It includes DNA replication and fate of DNA and its regulation and clinical significance.
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DNA metabolism
- 1. DNA Metabolism Prof (Dr) V. P. Acharya, MD, PhD
- 2. CENTRAL DOGMA The Flow of Information: DNA → RNA → Protein A gene is expressed in two steps: DNA is transcribed to RNA Then RNA is translated into protein proposed in 1958 by Francis Crick
- 3. Crick wrote….. These are the three transfers which the central dogma postulates never occur: Protein -> Protein Protein -> DNA Protein -> RNA CENTRAL DOGMA has been greatly misconstrued
- 4. DNA- major store of genetic information To transfer genetic information from parent to daughter cell, DNA must be duplicated. DNA duplication– DNA REPLICATION DNA→DNA Semi- conservative replication
- 5. Different models of DNA replication
- 7. Salient features of replication Each strand serves as a template/mould, over which a new complementary strand is synthesized Base-pairing rule is maintained Polymerization from 5’-3’ direction Old DNA is not degraded but conserved DNA polymerase synthesises a new complementary strand
- 8. Requisites for replication Substrates 4 deoxyribonucleotides; dNTP Template 2 separated DNA strands Enzymes Helicase DNA topoisomerases DNA polymerases DNA ligase RNA Primer
- 9. Steps of DNA replication Identification of origin of replication Unwinding of dsDNA to provide an ssDNA template Formation of replication fork Initiation of DNA synthesis and elongation Formation of replication bubbles with ligation of the newly synthesised DNA segments Reconstitution of chromatin structure
- 10. 3 stages of replication Initiation Elongation Termination
- 11. Initiation Replication starts with recognition of ori (origin of replication) Single site of ori– bacteria Multiple sites– mammals dnaA protein binds to ori ↓ Local denaturation & unwinding of an adjacent A-T rich region
- 13. dnaB / Helicase– Binds to ori and unwinds & separates the strands using ATP ↓ Replication fork formed Topoisomerases– Relieve supercoils Type I– Breaks one strand of DNA Type II– Breaks both strands Introduces negative supercoils SSB (Single strand binding protein)- Stabilizes the separated strands and prevents reannealing.
- 14. Action of Helicase Uses energy from ATP to unwind the duplex DNA SSB SSB SSB SSB
- 15. Mammals- SSB counterpart is RFA (replication factor) dnaC protein– reqd. for dnaB binding at ori dnaG / Primase– Complexes with proteins to form PRIMOSOME ↓ RNA primer synthesis
- 16. RNA Primer
- 17. RNA primer 10-200 nucleotides long Primase in prokaryotes synthesise it DNAP α in eukaryotes synthesise it Nucleophilic attack by 3’- OH group of the RNA primer on the phosphate of the first entering deoxynucleotide triphosphate
- 18. Replication fork
- 19. Components of replication fork DNA helicase Primase DNA polymerase SSB
- 20. Elongation After RNA primers laid down, 2 DNA polymerase III complexes attach Leading strand & lagging strand DNA chain which runs in 3’-5’ direction copied by DNAP III in 5’-3’ direction in a continuous manner Okazaki fragments on lagging strand DNAP I– removes RNA primers and fills in the gap between okazaki fragments Ligase– joins the segments
- 22. DNA replication is bidirectional Circular DNA
- 23. Okazaki fragments Short stretches of 150-250bp (eukaryotes) Found on lagging strand during DNA replication Later gap is filled by DNAP I and joined by DNA ligase enzymes Found in both eukaryotes and prokaryotes Approx. 250 per replication fork
- 24. DNA polymerase complex 3 bacterial and 5 eukaryotic DNAP
- 25. Properties of DNAP Chain elongation– 100 nucleotides/sec in mammals Proof reading-- rectification Processivity– How many nucleotides are to be added before it dislodges DNAP-III
- 26. Proteins involved in replication
- 27. Termination
- 28. ter binding protein – binds to the sequence ↓ prevents helicase from further action ↓ termination of replication
- 29. Which factor triggers DNA replication??? External signals are delivered to cells during the G1 phase of the cell cycle and activate the synthesis of cyclins Cyclins form complexes with cyclin- dependent kinases (CDK) Cascade of reaction Synthesis of S phase proteins like DNAP and thymidylate synthase
- 30. Eukaryotic replication Binding of origin recognition complex(ORC) to origins of replication during G1 phase ↓ ORC serves as a platform for highly complicated pre-RC complex formation ↓ Converted to RC by CDK and Dbf4- dependent kinase ↓ DNA polymerase α- primase complex synthesizes first primer
- 31. 2 characteristic features Histone complexes Telomeres- repeated end sequences of (TTAGGG)n and have typical sizes of 15– 20 kb at birth ↓ In somatic cells it is shortened after each cycle ↓ Germline and cancer cells have telomerases ↓ extend the 5′ end of lagging strands
- 32. Telomerase Reverse transcriptase Contains an RNA template Telomerase over-activity- cancer Under-activity- ageing
- 33. Modification after replication Methylation of DNA At C5 of C Catalysed by DNA methyl transferase Methylation occurs at G-C rich region of promoter sequence >90% methyl C s are in CpG dinucleotides Methylated areas- transcriptionally silent Aberrant methylation- cancer, ageing, ROS dependent damage
- 34. Difference between prokaryotic and eukaryotic replication Features Prokaryotes Eukaryotes RNA primer length ~50 nucleotides 9 nucleotides DNAP 3; I,II,III 5; α,β,γ,ε,δ Number of origin Single Many Okazaki fragments 1000-2000 nucleotides ~250 nucleotides Rate of replication ~500 nucleotides/sec ~100 nucleotides/sec
- 35. DNA replicates once and once only Pre-replicative sequence is formed- ORC-DNA complex with Cdc6 and Cdt1 at G1-S phase New nucleosomes are assembled behind the replication fork
- 36. Inhibitors of DNA replication Topoisomerase II (DNA gyrase) inhibitor Novobiocin– prevents ATP binding to gyrase Nalidixic acid & Ciprofloxacin- interfere with the breakage and rejoining of DNA chains Camptothecin– inhibits human topoisomerase I Nucleotide analogues– 2,3 dioxyinosine, Cytarabine Zidovudine, Acyclovir- terminate DNA chain elongation
- 37. Be like the stem cell-differentiate yourself from others
- 38. WWW.VPACHARYA.COM YOUTUBE CHANNEL V P ACHARYA HTTPS://WWW.YOUTUBE.COM/USER/ACHARYASONI1 For more ppt on Medical Biochemistry please visit my website
Editor's Notes
- This is not the first time that the idea of the central dogma has been misunderstood, in one way or another. In this article I explain why the term was originally introduced, its true meaning, and state why I think that, properly understood, it is still an idea of fundamental importance. This states that once "information" has passed into protein it cannot get out again.