Replication in Prokaryotes (E-Coli) PDF

Field of Medicine Medicine And Surgery Program Lecture: Replication (DNA Synthesis) in Prokaryotes Dr : George N. B. Morcos Date : 27 / 10 /2022  Let’s think together ▪ Why do you need to have an identical copy of a DNA?  ILOs By the end of the...

Field of Medicine Medicine And Surgery Program Lecture: Replication (DNA Synthesis) in Prokaryotes Dr : George N. B. Morcos Date : 27 / 10 /2022  Let’s think together ▪ Why do you need to have an identical copy of a DNA?  ILOs By the end of the lecture, the student should be able to: 1. Identify the proteins and enzymes involved in DNA replication. 2. Describe the synthesis of the leading and lagging strands.  Replication Definition: It is the synthesis of DNA strand using another strand as a template Function: Provide genetic information required by daughter cell from parent cell.  Replication Principles: It is semi-conservative  Enzymes of Replication 1. DNA Polymerases:  Enzymes of Replication 1. DNA Polymerases: They utilize deoxynucleoside triphosphates (dNTP) and remove pyrophosphate. DNA polymerases are template-directed enzymes read the parental nucleotide sequence in the 3` to 5` direction synthesize the new strand in 5` to 3` direction DNA polymerases require an RNA primer with a free 3`- hydroxyl group They cannot start from scratch by adding nucleotides to a free single stranded DNA template.  Enzymes of Replication 2. DNA Ligase: It joins ends of two segments of DNA by catalyzing the formation of phosphodiester bond.  Enzymes of Replication 3. DNA Helicase: It catalyzes unwinding of DNA double helix. The separation of DNA strands requires energy which is supplied by hydrolyzing ATP.  Enzymes of Replication 4. DNA Topoisomerases: They catalyze removal of supercoils that are formed during DNA unwinding. I. Topoisomerases I a) Breaks a phosphodiester bond in one DNA strand (produces a cut or nick), b) Allowing the intact strand to pass through the nick, c) Then it reforms the phosphodiester bond.  Enzymes of Replication 4. DNA Topoisomerases: II. Topoisomerases II Acts by making transient break in both DNA strands, release the supercoil and reseals the break. DNA gyrase a. Is a special type of topoisomerase II found in bacteria and plants. b. Has unusual ability to introduce negative supercoils to relaxed circular DNA using energy. N.B. a.Some anticancer agents target human topoisomerases I or II. b.Fluoroquinolones (Antimicrobial agents) target bacterial DNA gyrase.  Mention two characteristic properties of DNA polymerase in its action? Replication in Prokaryotes (E-Coli) Starts at a unique origin, Proceeds in opposite directions simultaneously. Form 2 replication fork. Replication of dsDNA is bidirectional, the replication forks move in both directions away from the origin  Replication in Prokaryotes (E-Coli) Steps I- Separation of the 2 DNA Strands & Formation of Prepriming Complex at the Replication Fork: A-Opening of DNA at Origin of replication 1. A unique origin rich in AT base pairs (origin of replication) (called ori C in E. coli) 2. dna A binds to ori C & produces local unwinding.  Replication in Prokaryotes (E-Coli) Steps I- Separation of the 2 DNA Strands & Formation of Prepriming Complex at the Replication Fork: B- Formation of Prepriming Complex at the Two Replication Forks A complex of dna B and dna C binds to Ori C dna B is a helicase that produces progressive unwinding of DNA double helix Single strand binding (SSB) proteins Bind cooperatively Stabilize the single strands (in their absence 2 stands can rewind again) Protect the single strand from nucleases  Replication in Prokaryotes (E-Coli) Steps II- Initiation of DNA Synthesis: Formation of RNA Primers RNA primer is formed by the action of primase, a DNA- dependent-RNA-polymerase. These primers are elongated by DNA polymerase III.  Replication in Prokaryotes (E-Coli) Steps III- Elongation: Synthesis of Both DNA Strands (Leading & Lagging Strands) 1- DNA polymerase III A. DNA-polymerase III synthesizes both strands of DNA ▪ Rate of elongation is from 20 to 50 nucleotides/ second. ▪ It is highly processive up to 50,000 nucleotides in one cycle.  Replication in Prokaryotes (E-Coli) Steps III- Elongation: Synthesis of Both DNA Strands (Leading & Lagging Strands) 1- DNA polymerase III B. Read the parental strand in the 3` to 5` direction (forms the new strand in the direction 5` to 3`) Leading strand Continuous manner In the direction of the advancing replication fork Lagging strand Discontinuously Opposite direction of the advancing replication fork In the form of Okazaki fragments (1000-2000 bases). Replication in Prokaryotes (E-Coli) Replication in Prokaryotes (E-Coli)  Replication in Prokaryotes (E-Coli) Steps III- Elongation: Synthesis of Both DNA Strands (Leading & Lagging Strands) 1- DNA polymerase III C. Proofreading of newly synthesized DNA strands: ▪ It hydrolytically removes the misplaced nucleotide (act as exonuclease) ▪ and replaces it with the correct nucleotide  Replication in Prokaryotes (E-Coli) Steps III- Elongation: Synthesis of Both DNA Strands (Leading & Lagging Strands) 2- DNA polymerase I It removes the RNA primers by its exonuclease activity then it fills the gaps between Okazaki fragments by DNA.  Replication in Prokaryotes (E-Coli) Steps III- Elongation: Synthesis of Both DNA Strands (Leading & Lagging Strands) 3- DNA ligase It joins the ends of adjacent fragments.  Replication in Prokaryotes (E-Coli) Steps III- Elongation: Synthesis of Both DNA Strands (Leading & Lagging Strands) 4- DNA polymerases I & II Are mostly involved in proofreading and DNA repair.  Replication in Prokaryotes (E-Coli) Steps III- Elongation: Synthesis of Both DNA Strands (Leading & Lagging Strands) 5- Topoisomerases Type I DNA topoisomerase Type II DNA topoisomerase  Why can’t DNA Polymerase III initiate the replication and it requires RNA primer?  Summary ▪ Enzymes of Prokaryotic DNA replication. ▪ Steps of Prokaryotic DNA replication.  References Lippincott's Illustrated Reviews: Biochemistry, 7th Edition. Chapter 30. https://www.youtube.com/watch?v=EYGrElVyHnU https://www.youtube.com/watch?v=2_-jSoSaaTA&t=52s https://www.youtube.com/watch?v=TnnlSVyRMJY&t=3s