BIOL10221 Molecular Biology Module 3 Lecture 1 - Genomes I 2024 PDF

BIOL10221 Molecular Biology Module 3 - Lecture 1: Genomes I Ray O’Keefe [email protected] What will we cover? Genome sizes and composition Role of replication origins Replication terminators and the role of Tus proteins in bacteria Why is it important? Multiple origins of...

BIOL10221 Molecular Biology Module 3 - Lecture 1: Genomes I Ray O’Keefe [email protected] What will we cover? Genome sizes and composition Role of replication origins Replication terminators and the role of Tus proteins in bacteria Why is it important? Multiple origins of replication are important for fast replication of larger genomes Bidirectional replication of circular DNA genomes must have a mechanism of stopping polymerases when they meet BIOL10221 Module 3 - Lecture 1 2 How are entire DNA molecules replicated? How is an organism’s whole genome replicated? What is a genome? the complete set of DNA molecules possessed by an organism BIOL10221 Module 3 - Lecture 3 1 The human genome comprises 3200 Mb DNA split into 24 linear DNA molecules shortest is 48 Mb longest is 250 Mb each one in a different chromosome In a normal diploid cell there are 46 chromosomes (Two sets of Chromosomes 1-22 and either XX or XY) 6400 Mb DNA! BIOL10221 Module 3 - Lecture 4 1 The E. coli genome comprises 4.64 Mb DNA contained in a single DNA this molecule is circular BIOL10221 Module 3 - Lecture 5 1 Replication of the E. coli genome begins at an origin of replication always the same position on the genome two replication forks - bidirectional BIOL10221 Module 3 - Lecture 6 1 Replication of human DNA also begins at origins of replication there are many on each chromosomal DNA molecule not always at the same sites each replication fork copies about 150 kb of DNA BIOL10221 Module 3 - Lecture 7 1 Initiation of E. coli DNA replication DnaA proteins bind close to the origin of replication the DNA becomes wound round these proteins BIOL10221 Module 3 - Lecture 8 1  DnaA proteins bind close to the origin of replication the DNA becomes wound round these proteins this forces the base pairs to break at the origin of replication origin is A-T rich (easier to come apart) base pairs are broken BIOL10221 Module 3 - Lecture 9 1 The prepriming complex is formed by attachment of DnaB proteins to the origin DnaB is a helicase DnaB breaks more base pairs so the replication forks move away from the origin Dna B helicase PREPRIMING COMPLEX BIOL10221 Module 3 - Lecture 10 1 The primosome is formed by attachment of two primase enzymes These make the RNA primers that initiate replication of the two leading strands Dna B helicase PREPRIMING COMPLEX Primase PRIMOSOME BIOL10221 Module 3 - Lecture 11 1 Now the events at the replication fork begin Primosome BIOL10221 Module 3 - Lecture 12 1 At the replication fork in E. coli Helicase (DNA B) breaks base pairs Single Strand Binding proteins (SSBs) protecting the bare single strands DNA topoisomerase unwinds the double helix preventing DNA supercoiling Primase making primers on leading & lagging strands DNA pol III (two copies) synthesizing DNA DNA pol I and DNA ligase removes primers and joins Okazaki fragments The gamma complex (clamp loader) attaches and detaches Pol III from the lagging strand Beta complex (sliding clamp) Holds Pol III onto template allowing it to slide BIOL10221 Module 3 - Lecture 13 1 At the replication fork in humans Helicase breaks base pairs Single Strand Binding proteins (SSBs) protecting the bare single strands DNA topoisomerase unwinds the double helix preventing DNA supercoiling Primase/DNA pol alpha making primers on leading & lagging strands DNA pol delta (two copies) synthesizing DNA FEN1 and DNA ligase removes primers and joins Okazaki fragments The ‘proliferating cell nuclear antigen’ (PCNA) sliding clamp that holds DNA Pol delta tightly onto the DNA BIOL10221 Module 3 - Lecture 14 1 The E. coli genome comprises 4.64 Mb DNA contained in a single DNA this molecule is circular BIOL10221 Module 3 - Lecture 15 1  Replication of the E. coli genome The replication forks always meet here Why? BIOL10221 Module 3 - Lecture 16 1 There are terminator sequences in the E. coli genome each one is the binding site for a Tus protein BIOL10221 Module 3 - Lecture 17 1 Tus proteins allow the replication fork to pass in one direction but not the other – Permissive face and Non-permissive face BIOL10221 Module 3 - Lecture 18 1 DNA strand separation followed by specific interaction of the Ter C6 base with the Tus lock domain results in replication fork arrest BIOL10221 Module 3 - Lecture 19 1 So the replication forks become trapped at the correct place BIOL10221 Module 3 - Lecture 20 1 Replication of human DNA also begins at origins of replication there are many on each chromosomal DNA molecule each replication fork copies about 150 kb of DNA forks just merge, don’t need exact control like in a circular DNA BIOL10221 Module 3 - Lecture 21 1 Resources Reading BROWN, Chapter 10 pages 201–207 ALBERTS (5th ed), Chapter 5 page 178-182, Chapter 6 page 201 and 205, 211-213 ALBERTS (6th ed), pages 187, 211, 215 Research paper https://www.nature.com/articles/nchembio.1857 PDF on Blackboard Blackboard PDF files with Objectives, Terms, Questions and Answers BIOL10221 Module 3 - Lecture 22 1  BIOL10221 Molecular Biology Module 3 - Lecture 1: Genomes I Ray O’Keefe [email protected]