2. Lecture 16 DNA Replication in Prokaryotes and Eukaryotes Rohini. K

3. Learning Objective 01 Nov 2011Rohini K FoM 2  An overview of the semiconservative mechanism of DNA replication  Initiation of replication; Elongation of DNA polymers  DNA polymerases  Telomeres

4. Central Dogma of Molecular Biology replication Reverse tran- scription Replication means making more copies of DNA. [Semi conservative mechanism produces 2 identical daughter DNA molecules]

5. Semi conservative replication. 2 strands of the parent molecules separated and each can serve as a template for the replication of a new complementary strand. This produces 2 daughter molecules and in each of the daughter molecule one parent strand and newly synthesized strand.

6. 1958: Matthew Meselson & Frank Stahl’s Experiment Equilibrium density gradient centrifugation

7. 1958: Matthew Meselson & Frank Stahl’s Experiment Semiconservative model of DNA replication

8.  After one generation -- only one band is seen but in the middle of the tube -- indicates both DNA synthesized are hybrid molecules.  After the 2 nd generation two bands seen ---- one in the middle and one in the top. --- indicates half of the DNA is light and half is hybrid DNA.  After 3 rd generation --75% of the DNA is light and 25% of the DNA is hybrid DNA.

9. Starting DNA Heavy/Heavy 1st generation All Heavy/Light 2nd generation Two Heavy/Light Two Light/Light 3rd generation Two Heavy/Light Six Light/Light

10. Thus Meselson and Stahl experiments proved the semi conservative mechanism of replication.

11. DNA SYNTHESIS [DNA Replication] Basic requirements for Replication: (a) Nucleotides—dATP, dGTP, dCTP,TTP (b) Template--Both strands of DNA (c) RNA primer-For initiating DNA synthesis (d) Enzymes- Helicases, Primase, DNA Polymerase Topoisomerase [ gyrase], and ligase. (e) Proteins– (i) Ori proteins. [DnaA,DnaB,DnaC] (ii) Single strand binding proteins

12. STEPS INVOLVED IN REPLICATION 1.DNA unwinding— Helicases with ATP catalyze the unwinding along with many proteins. 2. Primer synthesis – is catalyzed by primase 3. DNA synthesis—is catalyzed by DNA polymerase 4. Joining DNA fragments— ligase joins the end of newly synthesized DNA. 5. Super coiling control—DNA topoisomerase prevent tangling of DNA strand.

13. Replication: 1st step  Unwind DNA helicase enzyme  unwinds part of DNA helix  stabilized by single-stranded binding proteins single-stranded binding proteins replication fork helicase

14. INITIATION OF REPLICATION  Begins with Uncoiling of the DNA at a region called ORI [ A-T rich region]  ORI region --- ORI binding protein,[ Dna A, Dna B proteins] Rep protein, helicase and SSB protein.  Helicase --- attaches to the unwound DNA and continues the unwinding  Strand separation is maintained by single strand binding proteins and these allow the separated strands to serve as templates.

15. INITIATION – cont  DNA synthesis -- initiated by the synthesis of a short segment of RNA synthesized by the enzyme Primase.  RNA primer length [2-10nucleotides] 1. In prokaryotes  Single Origin of replication 2. In eukaryotes multiple site of replication

17. REPLICATION FORK RNA primer

18. ELONGATION PROCESS – DNA polymerase synthesis of 2 type of strands:  The two strands of DNA are antiparallel. [they run in opposite direction] From the initiation site [ORI], one parental strand runs in a 5’ 3’ while the other strand is running in the 3’ 5’ direction  DNA synthesis is always in the 5’ 3’ direction. Due to this 5’ 3’ polarity and antiparallel nature of DNA 2 different strands are formed. (1) Leading strand (2) Lagging strand

19. LEADING AND LAGGING STRAND  Leading strand uses 3’ 5’ parent strand as the template  The synthesis is continuous.  The direction of the synthesis coincides with the direction of replication.  Lagging strand uses 5’ 3’ parent strand as the template  The synthesis of the lagging strand is in fragments called the OKAZAKI fragments  These fragments are synthesized as the DNA opens up [separated].

20. DNA POLYMERASES  DNA polymerase III -- main replicating enzyme in bacteria. [ Pol III ]  DNA polymerase II -- repair and proof reading. [Pol II ]  DNA Polymerase I -- remove the RNA primers and replaces them with DNA. -- has proof reading activity. [Pol I ] Pol I has both 3’ to 5’ and 5’ to 3’ exonuclease activity

21. Eukaryotes have multiple DNA polymerases

22. Mammalian polymerases[eukaryotic] DNA polymerase AlphaDeltaEpsilonBetaGamma LocationNucleus Mitochon dria FunctionLagging strand synthesis Leading strand synthesis Repair of lagging strand Repair of leading strand Replicatio n Primase activity YesNo Yes 3 5’ exonuclease NoYes NoYes Proof reading activity Main enzyme - - 15%

24. ROLE OF LIGASE  The DNA Ligases ---- connects DNA Okazaki fragments during replication, ---- during repair and Recombination.  Newly replicated DNA is rapidly assembled into nucleosomes.

25. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Model of DNA replication

26. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Model of DNA replication

27. TELOMERE  End piece of the chromosome not replicated -- - TELOMERE.  Stability is lost If there is no mechanism to replicate telomere  The polarity is 5’ 3’  For initiating replication a RNA primer is required.  The RNA primer is removed by Pol I.  DNA Polymerase cannot fill the gap so a portion of template (3’ end ) is not replicated.

28. Repeating, non-coding sequences at the end of chromosomes  limit to ~50 cell divisions Telomerase  enzyme extends telomeres  can add DNA bases at 5 end  different level of activity in different cells  high in stem cells & cancers -- Why? telomerase Telomeres 5 5 5 5 3 3 3 3 growing replication fork TTAAGGG

29. TELOMERASE  Human telomere end consists of (TTAGGG) repeats which is highly conserved.  The shortening of telomere end is prevented by an enzyme Telomerase.  Telomerase acts like a reverse transcriptase. [ RNA]  In old age telomerase activity is lost leading to chromosome instability.  In cancer cells there is continued presence of telomerase activity leading to continued cell division..

30. Learning Outcomes  Explain the initiation of DNA replication.  Describe the formation of new DNA strands.  Explain the semi-conservative mechanism for DNA replication.  Compare and contrast DNA replication in prokaryotic and eukaryotic cells.  Explain the term ”telomeres”. 01 Nov 2011Rohini K FoM 29

31. References  Textbook of biochemistry: Lippincott’s Illustrated Reviews, 5th edition – Unit VI – Storage and Expression of Genetic Information.  Textbook of biochemistry: Vasudevan 5 th Edition - Chapter 41 – DNA Structure and Replication. 01 Nov 2011Rohini K FoM 30

32. 01 Nov 2011Rohini K FoM 31 Thank You