Dna Replication BMS 532 Lecture Notes PDF

DNA Replication BMS 532 BLOCK 3 LECTURE 2  Objectives 1. Summarize and Diagram a basic bidirectional replication fork from an origin of replication and summarize the steps of Replication from Origin of Replication through FLAP Endonuclease activity with particular emphasis on the molecules i...

DNA Replication BMS 532 BLOCK 3 LECTURE 2  Objectives 1. Summarize and Diagram a basic bidirectional replication fork from an origin of replication and summarize the steps of Replication from Origin of Replication through FLAP Endonuclease activity with particular emphasis on the molecules involved: Primase, RNA Primer, DNA polymerase (all forms discussed), Helicase, Ligase, Topoisomerase 2. Explain the process of origin firing and the role of multiple origins of replication in human replication and compare and contrast the activities and features of early and late firing origins of replication 3. Explain the process of origin licensing and firing and assess the consequences for alterations in the activity for the origin recognition complex, components of the pre-replication complex, and components of the pre-initiation complex (ORC, Pre-RC, Pre-IC) 4. Compare and contrast leading and lagging strand synthesis with emphasis on the molecules involved in each and the movement of the polymerase vs base addition 5. Outline the biochemical process that enables the addition of new nucleotides with emphasis on required biochemical functional groups and strand directionality and assess the consequence for lack of or loss of the 3’ hydroxyl group 6. Compare and contrast replication in circular dsDNA, in ssDNA (circular or linear), and linear dsDNA and Explain the cause of the limitation in the replication of linear chromosomes and the corresponding consequence (end-replication problem) 7. Compare and contrast types of polymerases in terms of their roles in the processes of replication and assess the consequence for errors in their activity 8. Compare and contrast topoisomerase I and topoisomerase II and assess the consequence for errors in their activity 9. Assess the consequences for replication and DNA sequence for errors in replication and in the activity of the replication machinery (corresponds to all slides) LO1 Basics of Replication Requires ACCESS to the DNA as well as the activation of the machinery Semi-conservative process means the machinery is relying on instructions to make the copy Replication is BIDIRECTIONAL from the origin Steps: ◦ Open DNA at the origin via helicase activity ◦ Stabilize strand and enable polymerase association (DNA polymerase requires dsDNA) and relieve strain upstream via topoisomerase activity ◦ DNA polymerase extension of strand by bringing new sugar backbone 5’ to 3’ ◦ Requires 3’OH group for extension of the strand ◦ Progression of replication is in the direction of helicase movement (direction of replication fork) ◦ This means one strand will have a polymerase playing “catch-up” as it can only add new bases 5’ to 3’ ◦ This creates fragments known as Okasaki fragments ◦ Remove primers and seal open gaps in the DNA backbone via DNA ligase (unite fragments) LO1  A Basic Replication Fork LO1, LO6 (Bacteria) LO1 DNA Replication at Replication Fork LO1 Eukaryotic Replication Forks LO1, LO6 Primers: Role in Replication Primer = short RNA segment complementary to DNA at origins of replication synthesized by PRIMASE Primer provides free 3’-OH which can be extended by DNA polymerase Okazaki fragments are also initiated by primers eventually replaced by DNA; DNA ligase joins ends LO2, LO3 Origins of Replication Formation of a replication “bubble” Average human chromosome = 150 million nucleotide pairs Replication fork moving along one chromosome from a single origin at 50 nucleotides per second = ~ 800 hours to complete DNA replication THUS, the efficient copying of the genome in larger genomes with multiple linear chromosomes requires multiple origins of replication LO2, LO3 Eukaryotic Chromosomes: Multiple Origins of Replication Replication units: replication origins activated in clusters in the genome LO2, LO3 DNA Replication and the Cell Cycle Replication is regionally specific ◦ Different origins initiate at different times during S phase Early vs. Late Replication regions are determined by different factors in different organisms ◦ Much of this area is under exploration and information is limited ◦ In yeast, specific sequences have been identified though not necessarily observed in other eukaryotes LO2, LO3 Late vs Early Replication DNA replication is not uniformly induced ◦ Cell type specific pattern = Replication Timing Program ◦ Established during G1 There are specific regions that replicate early and others that replicate late In general: ◦ Early = active gene transcription ◦ Late = lower gene transcription The origins of replication for these regions are distributed differently ◦ Early = between genes or at distinct locations ◦ Late = almost random Gnan et al 2020 LO2, LO3The Process of Initiation of Replication The 2 Stages of Replication Initiation (perspectives from yeast) 1. Origin Licensing 2. Origin Firing Explains the differences observed between early and late replicating regions Regulation and prevention of re-replication occurs by preventing origin re-licensing ◦ Pre-RC = pre-replication complex ◦ Contains Origin Recognition Complex (ORC) which promotes the binding of Cdc6, Cdt1, and MCM helicase ◦ Binding of MCM helicase initiates replication licensing ◦ Pre-IC = pre-initiation complex CRITICAL NOTE: Transcription impacts Replication and is a contributor to overall genomic instability Gnan et al 2020 LO2, LO3 LO2, LO3 LO2, LO3 LO1, LO4, LO5 DNA Synthesis Sugar backbone is comprised of phosphodiester bonds 3’OH is ESSENTIAL for synthesis ◦ Cleavage of the hydroxyl group stalls strand elongation Synthesis can only occur in 1 direction as we elongate at the 3’ OH (growing end) ◦ Thus the movement of polymerases along the template strand is limited ◦ Generates 2 types of synthesis ◦ Leading (polymerase moves seamlessly in same direction as the replication fork) ◦ Lagging (polymerase movement disjointed b/c in opposite direction of replication fork movement; Okazaki Fragments) LO1, LO6 Rolling Circle Replication Circular dsDNA One DNA strand is cut by a nuclease to produce a 3’-OH extended by DNA polymerase The newly replicated strand is displaced from the template strand as DNA synthesis continues Displaced strand is template for complementary DNA strand LO1, LO6 Single-Stranded DNA Virus Replication Rolling Circle Replication for circular Rolling Hairpin Replication for linear ssDNA ssDNA ◦ Host polymerases create initial dsDNA circle (1) Martin 2011 LO1, LO7 DNA Polymerases: Prokaryotes = 5 DNA pol I ◦ 5’ to 3’ exonuclease ◦ Serves to remove the RNA template during lagging strand synthesis ◦ Elongates the strand after removal of the RNA template until it reaches the next fragment DNA pol III ◦ Primary Molecule of ELONGATION ◦ Contains 2 of each of the following: ◦ The α (alpha) subunit = polymerase ◦ The ε (epsilon) subunit = 3’ to 5’ EXONUCLEASE; proofreading ◦ The θ (theta) subunit = stimulates proofreading ◦ The β (beta) subunit = sliding clamps for DNA; keep the polymerase bound ◦ The τ (tau) subunit = forms dimer for the α, ε, and θ subunits ◦ Contains 1 of each of the following: ◦ The γ (gamma) subunit = clamp loader for lagging strand synthesis; helps β subunits bind DNA ◦ The χ (chi) and Ψ (psi) subunits provide stabilization and connect the γ and τ subunits LO1, LO7 DNA Polymerases: Eukaryotes = 15 Pol α (alpha) ◦ Polymerase-primase capable of synthesizing the RNA primer (works with primase) ◦ Polymerase activity that elongates the primer with DNA nucleotides (~100) ◦ Capable of de novo DNA synthesis; lacks 3’ exonuclease activity Pol δ (delta) ◦ Main polymerase of lagging strand ◦ Can fill primer gap (displaces primer during fragment synthesis) Pol ε (epsilon) ◦ Main polymerase of leading strand; elongates the DNA strand Pol β (beta) and other pol ◦ DNA repair polymerases LO1, LO8 More on DNA Replication: Topoisomerases Movement of the replication fork is aided by topoisomerases ◦ Enzymes which unwind the DNA helix to permit strand separation ◦ Relieves torsional strain on the molecule Type I DNA topoisomerases unwind DNA by cutting one strand, rotating it around the second strand and then sealing the single strand break (nick) ◦ Only Type 1a topos can bind ssDNA ◦ Also only form found in all domains of life Type II Topoisomerases cut both strands LO1, LO6 The End-Replication Problem: Introduction to Telomeres The need for RNA primers and the limitations of DNA polymerases mean that not all parts of linear chromosomes can be properly replicated The ends of linear chromosomes cannot be fully replicated leading to progressive loss of information (End Replication Problem) Overtime, the loss of bases could significantly impact chromosome integrity and genes thus chromosomes have specialized structures to protect against loss Telomeres = Repetitive sequences of DNA at the ends of the chromosomes that assist with protecting the chromosome from genetic loss (along with other critical functions) LO1 Primer Removal: Pol Delta and Flap Endonuclease RNA primer must be removed as part of the finalization of the DNA Pol δ during Okazaki fragment synthesis causes displacement of RNA primer leading to a “flap” ◦ If the flap is too long, it must be processed by DNA2 (a nuclease/helicase) into a shorter flap The shorter flap is recognized and cleaved by Flap Endonuclease (FEN1) The backbone of the strand is sealed by DNA ligase uniting the fragments Questions Why do leading and lagging strand synthesis differ and in what way(s)? (LO1) Loss of which functional group prevents elongation during new strand synthesis? (LO5) What are the expected features for a section of DNA if it contains an origin of replication that is considered to be early firing? (LO2,LO3) What part of replication initiation is the same regardless of whether it is an “early” or “late” gene? What part is different? (LO2, LO3) Questions How is replication of dsDNA different from replication of ssDNA? (LO6) How are polymerases different across prokaryotes and eukaryotes? Across strands? (LO7) What is the role of topoisomerases in replication? (LO1 and LO8)