Dna replication in prokaryotes

For BSc Msc

1. DNA REPLICATION IN PROKARYOTES

2. DNA REPLICATION Contents Introduction DNA Structure Replication Model Enzymes involved in replication Mechanism of replication Replication in prokaryotes Conclusion Reference

3. What is replication? DNA replication is the biological process of producing two identical copies of   DNA  from one original DNA  molecule.

4. DNA replication occurs just before cell division(mitosis and meiosis). It occurs during interphase.

5. Synthesis Phase (S Phase) S Phase During interphase of the cell cycle. DNA Replication take place in the S Phase

6. DNA Structure DNA has three main components Deoxyribose (a pentose sugar) Base (there are different ones) phosphate

7. The Bases There are divided into two groups Pyrimidines and purines Pyrimidines (made of one six member ring) Thymine adenine Purine (made of six member ring, fused to a 5 member ring) cytosine Guanine

8. DNA Double Helix and Hydrogen Bonding Made of two strands of nucleotides that are joined together by hydrogen bonding Hydrogen bonding occurs as a result of complimentary base pairing Adenine and thymine pair up (2 bonds) Cytosine and guanine pair up(3 bonds) Each pair is connected through hydrogen bonding.

9. Modes of DNA Replication Conservative Semi-conservative Dispersive

10. Conservative • Both parental strands stay together after DNA replication. Semiconservative • The double-stranded DNA contains one parental and one daughter strand after replication. Dispersive • Parental and daughter DNA are interspersed in both strands after replication.

11. Enzymes of Replication DNA Replication requires these enzymes: DNA topoisomerase Helicases DNA Ligase SSB proteins RNA primase DNA polymerase

12. Helicases Helicase use energy from the ATP to break the hydrogen bond holding the base pair together. This allows the two parental strands of DNA to begin unwinding and form to replication forks.

13. 2. DNA topoisomerase Topoisomerases are enzymes that regulate the overwinding or underwinding of DNA . It relieve stress ahead of replication fork.

14. 3. SSB Protein (single stranded DNA binding protein) When DNA was unwinded into two single strand by helicase, it intend to form double stranded DNA and keep the single state of DNA in replication.

15. 4. RNA Primase Primase functions by synthesizing short RNA sequences that are complementary to a single-stranded piece of DNA, which serves as its template.

16. DNA ligase DNA ligases close nicks in the phosphodiester backbone of DNA. Biologically, DNA ligases are essential for the joining of Okazaki fragments during  replication.

17. 5. DNA Polymerase The enzyme responsible for DNA synthesis (replication) and repair is called DNA Polymerase.

18. DNA polymerase DNA polymerase I: It replace RNA primer with the DNA strand. DNA Polymerase II: Polymerase II has 3’ to 5’ exonuclease activities and participate in DNA repair. DNA Polymerase III: It is responsible for leading and lagging strand synthesis.

19. Steps of DNA Replication There are three steps involve in DNA replication. Initiation Replication begin at an the origin of the replication. Elongation New strands of DNA are synthesized by DNA Polymerase. Termination Replication is terminated differently in Prokaryotes and eukaryotes.

20. REPLICATION IN PROKARYOTES

21. DNA Replication in Prokaryotes (E.coli) The genome of E.coli is replicated bi-directionally and circular DNA. E.coli replicated is circular with no free ends. Replication of DNA in E.coli is also known as theta replication and it occurs in three steps. Initiation Elongation Termination

22. INITIATION Helicase unwinding DNA forming a replication fork. To initiate replication process multiple replicative proteins must assemble on these replication sites.

23. Elongation DNA polymerase III extends the RNA primer made by primase. All DNA polymerases make DNA in 5’-3’ direction. Two strand are formed. Leading strand Lagging strand

24. Leading strand synthesis On the template strand with 3’-5’ orientation, new DNA is made continuously in 5’-3’ direction towards the replication fork. The new strand that is continuously synthesized in 5’-3’ direction is the leading strand.

25. Leading & Lagging strand DNA polymerase III synthesizes DNA for both leading and lagging strands. After DNA synthesis by DNA pol III, DNA polymerase I uses its 5’-3’ exonuclease activity to remove the RNA primer and fills the gaps with new DNA .   Finally DNA ligase joins the ends of the DNA fragments together.

26. Lagging strand On the template strand with 5’-3’ orientation, multiple primers are synthesized at specific sites by primase (primosome complex) and DNA pol III synthesizes short pieces of new DNA (about 1000 nucleotides long) new DNA is in 5’-3’ direction.   These small DNA fragments that are discontinuously synthesizes are called Okazaki fragments (named after the discovered Reign Okazaki). The new strand which is discontinuously synthesized in small fragments is called the lagging strand.

27. Termination Once replication is complete, the two double stranded circular DNA molecules remain interlinked. Topoisomerase II makes double stranded cuts to unlink these molecules.

28. Conclusion Helicase uncoils the DNA RNA primase adds short sequences of RNA to both strands (the primer) The primer allows DNA polymerase III to bind and start replication DNA polymerase III adds nucleotides to each template strand in a 5'→3' direction These nucleotides are initially deoxyribonucleoside triphosphates but they lose two phosphate groups during the replication process to release energy One strand is replicated in a continuous manner in the same direction as the replication fork (leading strand) The other strand is replicated in fragments (Okazaki fragments) in the opposite direction (lagging strand)  DNA polymerase I removes the RNA primers and replaces them with DNA DNA ligase then joins the Okazaki fragments together to form a continuous strand