DNA Replication Lecture 7 PDF

BIOL2010 DNA replication Lecture 7 Dr M.L.Bellamy Overview 1) Composition/structure of DNA 2) DNA synthesis vs DNA replication 3) Problems and solutions in E.coli 4) The 3 steps in replication in E.coli 5) Differences in eukaryotes/viruses DNA replication St...

BIOL2010 DNA replication Lecture 7 Dr M.L.Bellamy Overview 1) Composition/structure of DNA 2) DNA synthesis vs DNA replication 3) Problems and solutions in E.coli 4) The 3 steps in replication in E.coli 5) Differences in eukaryotes/viruses DNA replication Steps Initiation→Elongation→Termination 1. How does replication start? 2. How does replication progress? 3. How does replication stop? DNA replication Eukaryotes Initiation Tightly linked to the cell cycle Initiation must only happen once per cycle Two step process ensures this DNA replication Eukaryotes Initiation Origins of replication arise from sections of DNA called Autonomously Replicating Sequences (ARS) Separated by ~30kb, so range from 10 on small chromosomes, to 1000s on the largest AT-rich consensus sequence (A region) in yeast is: 5'- /ATTTAYRTTT /A -3’ T T Y = any pyrimidine R = any purine DNA replication Eukaryotes Initiation/licensing (G1 phase) An Origin Recognition Complex of proteins (ORC) binds to the A region. DNA replication Eukaryotes Initiation/licensing (G1 phase) Accessory proteins (licensing factors) accumulate in the nucleus during G1. Cdc6 and Cdt1 bind to ORC. DNA replication Eukaryotes Initiation/licensing (G1 phase) Two helicases are loaded by Cdt1 onto dsDNA Cdc6/Cdt1 leave. HELICASE HELICASE (Mcm2-7) “Licensed” DNA replication Eukaryotes Initiation/activation (S phase) Pre-replication complex must be activated Additional proteins Active initiation complex/ Replisome progression complex DNA replication Eukaryotes Initiation overview G1 – Helicase loading; no helicase activation S – No helicase loading; helicases activated Replisome progression complex contains many proteins, including Replisome Replisome polymerases progression progression complex complex DNA replication Eukaryotes Elongation: Polymerases 5 main eukaryotic DNA polymerases: α, β, γ, δ, ε Polymerase α has its own primase activity, as well as polymerase, so can make its own primers It has no proofreading 3’-5’ exonuclease Not very processive (~20nt) since it does not associate with the eukaryotic sliding clamp protein called PCNA (proliferating cell nuclear antigen) Replication factor C (RFC) loads the PCNA onto the primer/DNA ready for a different Pol to bind DNA replication Eukaryotes Elongation: Polymerases 5 main eukaryotic DNA polymerases: α, β, γ, δ, ε Polymerase β is involved in repair Polymerase γ replicates mtDNA and has proofreading Polymerase δ (lagging strand) associates with PCNA and has proofreading Polymerase ε (leading strand) associates with PCNA and has proofreading DNA replication Eukaryotes Elongation: editing Pol δ can displace RNA primers, but can’t release them as rNMPs RNA "flap" produced by Pol δ Flap endonuclease-1 (FEN1) recognises 5’ flaps and cleaves off a short section, leaving a nick DNA ligase removes the nick N.B. primers can be mostly digested by RNAse H2, with 1 RNA nt left (like E.coli version). Then FEN1 removes last RNA nt. DNA replication Eukaryotes Elongation Not every pre- replication complex is used in each round of replication. Replication forks from one origin will pass through another origin (passive replication) ~5 × 104 origins for human-sized genome  DNA replication Eukaryotes Linear chromosomes The end replication problem Affects the lagging strand Last RNA primer may not be at the extreme 3’ end of the DNA→missed section 5’ 3’3 3’ 5’ 5 DNA replication Eukaryotes Linear chromosomes The end replication problem Even if it’s at the end, it will be removed because it’s RNA → ssDNA section 3’ 5’ 3’ 5’ DNA replication Eukaryotes Linear chromosomes The end replication problem 3’ 5’ n-10 strand Next replication Shortened strand is locked in Next replication Further end loss→gene shrinkage n-20 strand DNA replication Eukaryotes Telomeres – the end replication solution The DNA at the 3’ ends of chromosomes doesn’t encode genes Instead it is multiple repeats (100s-1000s) of a simple sequence: TTAGGG The last 20-200nt (species dependent) at the 3’ end are ssDNA made of these repeats DNA replication Eukaryotes Telomeres – the end replication solution Once the telomeric DNA is gone (~40 replications)→gene loss →cells stop dividing, a point called the Hayflick limit DNA replication Eukaryotes How do embryonal cells, cancer cells, stem cells and other types divide more times than the Hayflick limit? → Synthesis of new telomeric DNA Telomerase is a ribonucleoprotein It contains an RNA strand (450 nt) which has the sequence 5’CUAACCUAAC 3’ This acts as a template for the synthesis of new telomeric repeats, growing the telomere DNA replication Eukaryotes Telomerase – the end replication solution Telomerase binds and extends ssDNA Pol α (primase) binds and adds a primer Pol δ binds primer and extends it DNA ligase fills in nick DNA replication Eukaryotes Mitochondrial DNA 2-10 copies of circular mtDNA per mitochondrion Unidirectional replication (1 fork) Pol γ synthesizes leading strand Lagging strand is RNA Okazaki fragments DNA replication Viruses Bacteriophages Small circular genome Some use a “rolling circle” mechanism to continuously synthesize new DNA Sigma (σ) replication Nick created Nick acts as Pol displaces existing primer for Pol strand (unrolls it) DNA replication Viruses RNA genomes Many animal and plant viruses have genomes composed of RNA Have an RNA-dependent RNA polymerase called RNA replicase encoded by the viral genome The plus strand RNA is copied directly to make the minus strand, which is used as a template for making more plus strand Can be self-priming – no primer required No proof-reading → highly error prone DNA replication Viruses Retroviruses e.g. HIV RNA genome Virally encoded reverse transcriptase creates a DNA strand using RNA as template and tRNALys as a primer RNA half is degraded by RNase H Second DNA strand synthesized using first as template→Incorporated into host genome DNA replication Conclusions 1. DNA synthesis is simple 2. DNA replication is complex 3. Principles, enzymes and steps are surprisingly similar across all levels of life