DNA Synthesis

1. DNA Synthesis Lecture 31 MukundModak, Ph.D.

2. DNA Objectives: • Define a DNA polymerase reaction and its components. • Where does DNA replication begin? Where does it terminate? • Know about leading and lagging strand synthesis. • How many primers are required for each strand synthesis? • What are Okazaki fragments? Remember some of the components required in lagging strand synthesis such as Pol I to remove RNA primers and DNA ligase to join the Okazaki fragments. • Know the polarity of DNA to be replicated. • Know the major eukaryotic DNA polymerases. • Remember clamp protein( PCNA in eukaryotes and B clamp in prokaryotes) that clamps DNA pol ( mainly delta and epsilon) to template and speeds up polymerase rate. • What does enzyme telomerase do? It has RNA component with sequence complimentary to telomeric( DNA) sequences.

3. Watson-Crick Model Base 3’ 2’ • Double helix • Antiparallel • Base-paired • Base stacking • Major groove • Minor groove • Sugar-phosphate • Backbone • Helix structure by X-ray diffraction O P Base O 5’ O P Base Postulations: Semi conser- vative replication via base pairing principle. Expression via transcription (by same base pairing rule) O 5’ 3’

4. Review of Basics • DNA replication is semiconservative

5. Review of Basics • DNA is synthesized by the addition of a deoxynucleotide to the 3 end of a polynucleotide chain • Base-pairing between incoming dNTP and template strand provides specificity

6. Antiparallel Double Strand 5’ 3’ 3’ 5’ Base Polymerization reaction O 5’ OH + 5’ PPP = 3’O - P - 5’O + PPi 3’ P-P-P-O-H2C Therefore chain extends: 3’ to 5’ Overall growth: 5’ to 3’ 3’ OH Base O 5’ 5’ 3’ P-P-P-O-H2C OH 5’P New chain 3’ 5’P OH 3’ 5’ OH

7. DNA dependent DNA polymerases are key enzymes for DNA synthesis Requirements: Template, primer, 4dNTPs, Mg 2+  Nucleotidyl transferase reaction  Template dependent dNTP selection  direction of synthesis: 5’  3’ Imp: Primer (DNA or RNA) with 3’OH, a minimum of 10 bases hydrogen bonded to template 5’ 3’ 3’OH 3’OH 3’OH 5’ 5’ 5’ Enzyme Mg.dNTP Primer n+1 + PPi Prokaryotes contain 3 DNA polymerases: I, II and III

8. Specialized enzymes and factors • Specialized polymerases that synthesize primers and DNA • Editing exonucleases to work with polymerases. • Topoisomerases that convert supercoiled DNA to relaxed form • Helicases that separate two parental strands of DNA • Accessory proteins promoting tight binding of polymerase to DNA and • thereby increase the speed of polymerases (sliding clamps) • Details of the process

12. Leading vs Lagging Strand Replication 3’

15. DNA REPLICATION PROTEINS dnaA : Ori C dnaB : begins unwinding (helicase) Rep Helicase : Ongoing unwinding SSB : Stabilize ss DNA Gyrase and Topoisomerase : Supercoil to relax transition Primase : Synthesis of primer (RNA) Pol III complex : DNA synthesis Pol I : Primer removal and gap filling DNA ligase : Join DNA ends Topo IV : Decatenates replicated circles Topo IV

16. REPLICATION FORK SUMMARY Begins with unwinding of two strands with opposite polarity Two replisome assemblies (Primase + Pol III complex) Leading strand (continuous synthesis) Lagging strand (discontinuous synthesis) a. Looping of the strand to transiently change polarity and permit primer synthesis b. DNA synthesis in pieces (Okazaki fragments) c. Pol I to remove RNA primers and replace by DNA d. DNA ligase to join small DNAs into a single large molecule with ATP or NAD as a cofactor Termination signal: TUS factor Two forks reach each other at mid point of the circle and are stalled by TUS factor Replication completes with generation of catenated circles. Seperation of circles by Topo IV

17. PROOF READING ACTIVITY OF POL I (3’ to 5’ exonuclease activity)

18. Structure of first DNA polymerase (Klenow Fragment of E.coli pol I, 1985) Nomenclature of various structural units (1992) Thumb Palm Fingers 3’ – 5’ exonuclease The crystal structures of numerous DNA polymerases solved later, showed same general anatomy

19. Lecture 8

20. Eukaryotic DNA replication G0, G1, S and M Phases (DNA replication in S – phase) Many polymerases and accessory factors required Multiple initiation points to replicate (3 billion bps) Linear chromosome Overall replication scheme similar to prokaryotes Problem with 5’ – RNA primer removal and fill up Solution to this problem is telomerase action

21. Eukaryotic DNA Polymerases • a : Repair and Replication and primase function • : Repair function g : Mitochondrial DNA polymerase d : Replication with PCNA (processivity factor) • : Replication к: Repair function i: Repair function Telomerases Terminal deoxynucleotidyl transferase Viral reverse transcriptase Viral replication

22. Eukaryotic Fork • Three polymerases required for replication • Pol synthesizes primer RNA and very little DNA (non-processive, no proofreading exonuclease) • Pol and Pol both are processive (interact with PCNA) and have proofreading exonucleases

23. Important Participants In Eukaryotic DNA Replication • Pol (alpha) (delta) and (epsilon) primer synthesis and DNA synthesis (There is a separate primase in prokaryores) • Sliding clamp or processivity factor: PCNA Increases rates and length of DNA (Equivalent to β clamp in prokaryotes) • FEN-1 nuclease removes RNA primers (In prokaryotes, 5’- nuclease activity of pol I does it) • RPA is a single strand binding protein (SSB in prokaryotes)

24. 5 3 5 3 End-Replication Problem 5 3 + 5 3 Process Okazaki Fragments 5 3 + 5 3

25. Telomere Structure • Telomeres composed of short (6-10 bp) repeats (2000 – 3000) • G-rich in one strand, C-rich in other • TTAGGG/CCCTAA 5 3 G-rich C-rich

27. TERMINAL DEOXYRIBONUCLEOTIDYL TRANSFERASE (TdT) 5’ 3’ (ds DNA) Or (ss DNA) TdT 3’ 5’ DNAn+1 + PPi dNTP + Mg2+ 5’ 3’ • Addition of dNTPs on to 3’ OH end of DNA • No template requirement • No template guidance but will extend 3’OH ends of ss or ds DNA • with all 4 dNTPs Biological novelty • Found in only vertebrate species • In preimmunocytes (thymus) • Absent in mature circulating lymphocytes • Used as a lympho blastic leukemia marker • Involved in generation of diversified antibody genes

28. AZTTP as a Chain Terminator