Dna replication prokaryotes

PROKARYOTIC DNA REPLICATION

1. DNA REPLICATION IN PROKARYOTES DR. ANU P. ABHIMANNUE, ASSISTANT PROFESSOR, DEPARTMENT OF BIOTECHNOLOGY, ST. MARY’S COLLEGE, THRISSUR

2. REPLICATION Prokaryotic  DNA Replication is the process by which a prokaryote duplicates its DNA into another copy that is passed on to daughter cells DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 2

3. SCHEMES OF REPLICATION DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 3 Three types of DNA replication methods were postulated Conservative method Semi- Conservative method Dispersive method

4. CONSERVATIVE METHOD In conservative replication, the original strands would remain together as would the two newly synthesized strands. Hence, one of the daughter duplexes would contain only parental DNA, while the other contain only newly synthesized DNA. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 4

5. SEMI-CONSERVATIVE METHOD Semi-conservative mode was suggested by Watson and Crick in 1953. The daughter duplexes consist of one complete strand inherited from the parental duplex and one complete strand that has been newly synthesized . It is said to be semiconservative because each daughter duplex contains one strand from the parent structure. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 5

6. DISPERSIVE METHOD DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 6 The parental strands would be broken into fragments, and new strands synthesized in short segments, which further join together to form a complete strand. As a result, the daughter duplexes would contain strands that were composites of old and new DNA.

7. POSSIBILITIES O DNA REPLICATION DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 7

8. EVIDENCE To gain evidence on DNA replication, Studies on bacteria was conducted by Matthew Meselson and Franklin Stahl of the California Institute of Technology in 1957. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 8 Franklin Stahl : Born October 8, 1929 ),  American molecular biologist and geneticist.  Matthew Meselson : Born May 24, 1930, American geneticist  & molecular biologist 

9. PRINCIPLE They used heavy (15N) and light (14N) isotopes of nitrogen to distinguish between parental and newly synthesized DNA strands. The density of a DNA molecule is directly proportional to the percentage of 15N or 14N atoms it contains. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 9

10. Procedure DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 10

11. https://www.mun.ca/biology/scarr/Meselson_StahL_experiment.html 11

12. EQUILIBRIUM DENSITY-GRADIENT CENTRIFUGATION Extracted DNA was mixed with a concentrated solution of CsCl and centrifuged to equilibrium at high speed in an ultracentrifuge. Cesium ions form a density gradient with the lowest density of Cs at the top of the tube and greatest concentration at the bottom. During centrifugation, DNA fragments within the tube become localized at a position having a density equal to their own density, which in turn depends on the ratio of 15N/14N. The greater the 14N content, they localize higher in the tube. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 12

13. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 13

14. CONCLUSION The appearance of a hybrid band and the disappearance of the heavy band after one generation eliminates conservative replication. The subsequent appearance of two bands, one light and one hybrid, eliminates the dispersive scheme . As long as replication continued semi-conservatively , the original heavy parental strands remain intact and occupied a smaller percentage of the total DNA. Hence, DNA replication was found to be semi-conservative in nature . DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 14

15. PROKARYOTIC DNA REPLICATION In 1963,  John Cairns reported the process of replication in  E . coli  bacteria by autoradiography . The replication process can be broadly divided into 3 sections Initiation Elongation Termination DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 15 Cairns: (21 November 1922 – 12 November 2018 ); British physician and molecular biologist

16. INITIATION

17. ORIGIN Replication begins at a specific site on the chromosome called origin. Origin of replication of E . coli is called oriC It is of 245 bp length, where a number of proteins bind for initiation. It is a conserved sequence in prokaryotes, rich in AT residues. Ori C consists of two types of sequences 13mer - three repeats of 13bp 9mer - five repeats of 9bp DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 17

18. PROTEINS IN INITIATION DnaA and DnaC : proteins helping in the recognition of the specific sequence at oriC Helicase : Enzyme, in presence of ATP unwinds DNA by breaking hydrogen bonds between the nitrogenous base pairs to form replication forks  . Single-strand binding proteins:  coat the single strands of DNA near the replication fork to prevent them from winding back into double helix. DNA gyrase : Type II topoisomerase enzyme, relieves the mechanical strain that builds up during replication by removing positive supercoils DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 18

19. HELICASE E. coli has 12 different helicases. One specific helicase ie , DnaB helicases serves as the major unwinding enzyme during replication. DnaB helicase consists of six subunits arranged to form a ring-shaped protein that encircles a single DNA strand. It is first loaded onto the DNA at the origin and translocates in a 5´ → 3´ direction along the lagging-strand template, unwinding the helix as it proceeds.

20. DNA GYRASE It is a type II topoisomerase enzyme, change the state of supercoiling in DNA molecule . Moves along the DNA ahead of replication fork, removing positive supercoils . Mechanism: Cleaves both strands of the DNA, passing a segment of DNA through the break to the other side, and then seals the cuts. It require ATP hydrolysis DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 20

21. INITIATION Dna A protein with ATP, binds to the  9mer  sequences of  oriC . This binding allows the opening of  13mer  sequences of  oriC  by bending the DNA . Helicase bind to  13mer  repeats which are recognized by DnaC . Once helicase is settled on  oriC  (at  13mer ) the DnaC protein will be released . Helicase unwinds the DNA duplex into a Y – shaped structure called replication fork Meantime, tension developed is relieved by DNA gyrase DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 21

22. REPLICATION FORK The  replication origin forms a Y shape, and is called a  replication fork . The two replication forks move outwards in opposite directions that is, bi directionally At the end they meet at a point across the circle from the origin, where replication is terminated. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 22

23. ELONGATION DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 23

24. Enzymes DNA pol III – Major enzyme required for DNA synthesis . Primase - Synthesize a short segment of RNA called primer DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 24

25. DNA POLYMERASES Pioneer work in the study of DNA Pol was carried out by Arthur Kornberg at Washington University in the 1950s It adds nucleotides to the growing DNA chain that is complementary to the template strand. For polymerization to proceed , the enzyme need DNA and energy from all four dNTPs . DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 25 Arthur Kornberg  (March 3, 1918 – October 26, 2007) American biochemist   who won the Nobel Prize in Physiology or Medicine  1959

26. TYPES OF DNA POL In 1969, studies on a mutant strain of E. coli revealed that apart from Kornberg enzyme (DNA polymerase I), several distinct DNA polymerases are present. A typical bacteria contains 300 to 400 molecules of DNA polymerase I but only about 10 copies of DNA polymerase III. In prokaryotes, three main types of polymerases are known: DNA pol I - accessory enzyme in DNA replication DNA pol II - along with DNA pol I, is required for DNA repair. DNA pol III – Major enzyme required for DNA synthesis. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 26

27. DNA POLYMERASE I DNA polymerase I is considered as three different enzymes in one as it has 3 different functions. 5´ → 3´ exonuclease activity can degrade RNA stretches created by primase in lagging strand. 5´→ 3´ polymerase activity that fills the resulting gap in lagging strand 3´→5ˊ exonuclease activity DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 27

28. DNA POL STRUCTURE The holoenzyme consist of 10 different subunits Two core polymerases which replicate the DNA, Two or more clamps , which allow the polymerase to remain associated with the DNA , A clamp loading complex, which loads each sliding clamp onto the DNA. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 28

29. DNA POL STRUCTURE - CLAMP Clamp – non - catalytic component of the holoenzyme for keeping polymerase associated with the DNA template . Doughnut-shaped clamp encircles DNA and provides 2 contrasting properties for the enzyme: long stretch association of enzyme with template loose attachment with the template for easy movement. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 29

30. DNA POL STRUCTURE – CLAMP LOADER Clamp loader – multi subunit of enzyme containing two t subunits, which hold the core polymerases Clamp loading complex in ATP-bound state assembles at primer-template junction for holding the clamp. Once DNA moves through the opening in the clamp wall, ATP bound to the clamp loader is hydrolyzed, causing the release of the clamp, which closes around the DNA. The clamp is then ready to bind polymerase III. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 30

31. LIMITATIONS OF DNA POLYMERASE Two important restrictions: DNA Pol add Nucleotides only in the 5′ to 3′ direction. Enzyme requires a free 3′-OH group to add nucleotides, so, if a free 3′-OH group is not available. Then how does it add the first nucleotide? DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 31

32. PRIMER A short segment of RNA can provide the required 3´ OH terminus for DNA Pol and is called a primer. This 3 ´ OH carries out a nucleophilic attack on the 5´ phosphate of the incoming Nucleoside TriPhosphate . Primer is five to ten nucleotides long and complementary to template DNA. Primer is synthesized by RNA polymerase known as primase DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 32

33. PRIMOSOME Primosome is a combination of primase and helicase enzymes. Helicase moves along the lagging-strand template processively ( i.e., without being released from the template strand during the lifetime of the replication), thereby opening the duplex. Primase periodically binds to the helicase and synthesizes primers. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 33

34. REPLISOME Replisome refers to the entire complex of proteins that are active at the replication fork. Complex includes DNA polymerase III holoenzyme , the helicase, SSBs and primase . DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 34

35. LEADING STRAND Strand complementary to 3′ to 5′ parental DNA strand synthesized continuously in a single stretch Synthesized towards the replication fork Synthesized in 5′to3′ direction. LAGGING STRAND Strand complementary to 5′ to 3′ parental DNA Synthesized discontinuously , in fragments -  Okazaki fragments. Synthesized away from the replication fork. Synthesized in 3 ′ to 5′ direction DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 35

36. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 36 https://www.netclipart.com/isee/xmoTbT_dna-structure-clipart-dna-replication-enzymes-unzip-the/

37. OKAZAKI FRAGMENTS Short fragments on the lagging strand is known as Okazaki fragments. Discovered by Reiji Okazaki of Nagoya University, Japan. Synthesis of O kazaki fragments require individual primer. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 37 October 8, 1930 – August 1, 1975, Japanese  molecular biologist

38. After the synthesis of leading and lagging strand, the polymerase is detached from the site of replication . Thus bringing an end to elongation DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 38

39. Termination DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 39

40. ENZYMES RNase H – An endogenous enzyme that cleaves the RNA strand of an RNA–DNA duplex in lagging strand. DNA polymerase I – exonuclease activity, removing primers DNA ligase – Joining of DNA fragments DNA topoisomerase II – aids dissociation of 2 different circular DNA DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 40

41. DNA LIGASE DNA ligase joins Okazaki fragments into a continuous strand. It facilitates the joining of DNA strands by catalyzing the formation of a phosphodiester bond Catalyze 2  covalent phosphodiester bonds between  3‘OH end and 5 ' phosphate end of nucleotides. Two ATP molecules are consumed for each phosphodiester bond formed . DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 41

42. TERMINATION DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 42

43. PROOF READING Incorrect nucleotides are often removed by DNA polymerase I during termination of replication. The enzyme directs into the 3ˊ → 5ˊ exonuclease action that removes mismatched nucleotide . This activity removes 99 out of every 100 mismatched bases. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 43

44. TERMINATOR RECOGNIZING SEQUENCES At the last stage of termination, two replication fork meets at terminator recognizing sequences, called as a  Ter . Ter  sequences with TUS protein create a complex which arrests the replication fork. At this complex, the process of replication is completed and all other proteins and enzymes leave this site . Only DNA topoisomerase II remains in action, it cuts both strands, dissociates Ter -TUS complex and two different circular DNA is generated DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 44

45. RATE OF REPLICATION The replication of an entire bacterial chromosome in approximately 40 minutes at 37°C requires that each replication fork move about 1000 nucleotides per second. DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 45

46. REFERENCES Gerald Karp (2010). Cell and molecular biology: concepts and experiments (6 th ed.). John Wiley & sons. ISBN-13 978-0-470-48337-4. https :// www.mun.ca/biology/scarr/Meselson_StahL_experiment.html https://geneticeducation.co.in/prokaryotic-dna-replication / DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR . 46