Replication in Prokaryotes (E-Coli) PDF

Field of Medicine Medicine And Surgery Program Lecture: Replication (DNA Synthesis) in Prokaryotes Dr.Hidi Azmy El Tahawy Date : 22 / 10 /2024  Let’s think together ▪ Why do you need to have an identical copy of a DNA?  ILOs By the end of t...

Field of Medicine Medicine And Surgery Program Lecture: Replication (DNA Synthesis) in Prokaryotes Dr.Hidi Azmy El Tahawy Date : 22 / 10 /2024  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: Process of duplication of the entire DNA occur before cell division. So each daughter cell gets a complete copy. Function: Provide genetic information required by daughter cell from parent cell.  Replication Principles: It is semi-conservative: Each strand serves as a template for a new strand, the new duplex is ½ parent template & 1/2 new DNA  Replication DNA template Free nucleotides Requirments Enzymes ATP Mechanism of DNA Replication Initiation - Proteins bind to DNA and open double helix - Prepare DNA for complementary base pairing Elongation - Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA Termination - Proteins release the replication complex  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 1. DNA Polymerases:  Enzymes of Replication 2. DNA Ligase: It joins ends of two segments of DNA by catalyzing the formation of a phosphodiester bond.  Enzymes of Replication 3. DNA Helicase: It catalyzes the 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 the 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. d) Need no ATP.  Enzymes of Replication 4. DNA Topoisomerases: II. Topoisomerases II Acts by making a transient break in both DNA strands, releasing the supercoil and resealing the break. Need ATP. DNA gyrase a. Is a special type of topoisomerase II found in bacteria and plants. b. Has an 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 bi-directional; the replication forks move in both directions away from the origin Steps: I- Separation of the 2 DNA Strands 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. Steps: II-Formation of Prepriming Complex at the Replication Fork: A complex of dna B and dna C binds to Ori C dna B is a helicase that produces progressive unwinding of the DNA double helix. Single-strand binding (SSB) proteins Bind cooperatively. Stabilize the single strands (in their absence 2 stands can rewind). Protect the single strand from nucleases. Steps: III- Initiation of DNA Synthesis: a-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. Steps: III- Elongation: B-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. Steps: III- Elongation: B-Synthesis of Both DNA Strands (Leading & Lagging Strands) 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) Steps: III- Elongation: B-Synthesis of Both DNA Strands (Leading & Lagging Strands) C. Proofreading of newly synthesized DNA strands: ▪ It hydrolytically removes the misplaced nucleotide (acts as an exonuclease). ▪ and replaces it with the correct nucleotide. Steps: III- Elongation: B-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. Steps: III- Elongation: B-Synthesis of Both DNA Strands (Leading & Lagging Strands) 3- DNA ligase It joins the ends of adjacent fragments. Steps: III- Elongation: B-Synthesis of Both DNA Strands (Leading & Lagging Strands) 4- DNA polymerases I & II Are mostly involved in proofreading and DNA repair. Steps: III- Elongation: B-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