BIOL10221 2024 DNA Replication II Lecture Notes PDF

BIOL10221 Molecular Biology Module 2 - Lecture 2: DNA Replication II Ray O’Keefe [email protected] What will we cover? Template dependent DNA synthesis during replication DNA polymerases – functions and properties Leading and Lagging strand DNA replication at the re...

BIOL10221 Molecular Biology Module 2 - Lecture 2: DNA Replication II Ray O’Keefe [email protected] What will we cover? Template dependent DNA synthesis during replication DNA polymerases – functions and properties Leading and Lagging strand DNA replication at the replication fork Okazaki fragments and their joining Why is it important? By using a template, DNA can be replicated (copied) exactly without any errors DNA polymerases are the cellular enzymes that can make DNA strands from nucleotides Mutations in DNA polymerases can cause colorectal cancer DNA replication can only occur in one direction but the two strands at the replication fork are in opposite orientations BIOL10221 Module 2 - Lecture 2 2 DNA replication occurs by the semiconservative method DNA topoisomerases nick/cut the DNA to relieve the stress of DNA double-helix unwinding Helicases break the base pairs The replication fork progresses replication fork along the DNA molecule BIOL10221 Module 2 - Lecture 3 2 How are the new strands of DNA synthesized at the replication fork? The process is called template- dependent DNA synthesis The template is DNA, so this is DNA-dependent DNA synthesis BIOL10221 Module 2 - Lecture 4 2 DNA-dependent DNA synthesis is carried out by an enzyme called a DNA-dependent DNA polymerase DNA synthesis is always in the 5´3´direction This requires a primer to initiate synthesis of the new strand BIOL10221 Module 2 - Lecture 5 2 Without a primer there is no DNA synthesis BIOL10221 Module 2 - Lecture 6 2 Most DNA polymerases can also degrade DNA this is called the exonuclease activity there are two possible types of exonuclease activity 3´5´exonuclease activity the polymerase can remove nucleotides it has just inserted this is called proofreading – allows errors to be corrected BIOL10221 Module 2 - Lecture 7 2 Most DNA polymerases can also degrade DNA this is called the exonuclease activity there are two possible types of exonuclease 5´3´exonuclease activity the polymerase can remove DNA already attached to the template we will come back to this later BIOL10221 Module 2 - Lecture 8 2 α – alpha δ - delta BIOL10221 Module 2 - Lecture 9 2 DNA synthesis BIOL10221 Module 2 - Lecture 10 2 What happens at the replication fork? First, the separated single strands must be protected if not they may just re-attach to one another or they might be attacked by nucleases The strands are protected by single-strand binding proteins or SSBs Example in eukaryotes Replication protein A BIOL10221 Module 2 - Lecture 11 2 One strand can be copied by continuous DNA synthesis This is called the leading strand Copying of this strand requires a primer primer BIOL10221 Module 2 - Lecture 12 2 The primer is made of RNA in bacteria the primer is made by the primase enzyme the primer is 4-15 nucleotides in length once the primer has been made, DNA pol III makes the new strand BIOL10221 Module 2 - Lecture 13 2 In eukaryotes the process is slightly different The RNA primer is first extended by DNA pol alpha DNA pol alpha adds about 20 nucleotides then DNA pol delta makes the rest of the new strand BIOL10221 Module 2 - Lecture 14 2 How is the lagging strand copied? DNA synthesis is always in the 5´3´direction the lagging strand must therefore be copied from a primer placed at the replication fork. primer BIOL10221 Module 2 - Lecture 15 2  What happens when the replication fork moves? The leading strand is extended by more DNA synthesis But the lagging strand has to be made in sections BIOL10221 Module 2 - Lecture 16 2 The sections are called Okazaki fragments These must be joined together and RNA primers removed to complete lagging strand synthesis BIOL10221 Module 2 - Lecture 17 2 Discovery of Okazaki Fragments Added radioactive nucleotides to Bacteria for different amounts of time Separated DNA by size using Centrifugation Small DNA fragments only seen at shorter timepoints BIOL10221 Module 2 - Lecture 18 3 Joining two Okazaki fragments in Bacteria BIOL10221 Module 2 - Lecture 19 2 α – alpha δ - delta BIOL10221 Module 2 - Lecture 20 2 Joining two Okazaki fragments in Eukaryotes FEN1 is an endonuclease BIOL10221 Module 2 - Lecture 21 2 Resources Reading BROWN, Chapter 10 pages 194–208 ALBERTS, Chapter 6 pages 205-210 Blackboard PDF files with Objectives, Terms, Questions and Answers BIOL10221 Module 2 - Lecture 22 2  BIOL10221 Molecular Biology Module 2 - Lecture 2: DNA Replication II Ray O’Keefe [email protected]