The Central Dogma DNA structure and DNA replication DNA replication (continued) RNA Synthesis Protein synthesis DNA – an emblem of the 20th century. 1.! A simple yet elegant structure – a double helix with a sugar phosphate “backbone” linked to 4 types of nucleotide on the inside that are paired according to basic rules. Amazingly this simple molecule has the capacity to specify Earth’s incredible biological diversity. 2.! The double-stranded structure suggests a mode of copying (replication) 3.! The “strings” of the 4 bases are a digital code that specifies life. Prof. David McConnell Smurfit Institute of Genetics These lectures cover the research that led to the elucidation of the replication (copying/ reproduction) of DNA, and how DNA can generate protein products. Summary of Lecture 1 1.! DNA is a double stranded helix (Watson and Crick 1957) 2.! The strands are anti-parallel: 5’ to 3’ and 3’ to 5’ 3.! The two strands are held together by base pairs: A=T and G=C. 4.! The strands have complementary base sequences. 5.! The structure of DNA is independent of base sequence (not quite true) 6.! DNA replication is semi-conservative (Meselson-Stahl, 1958) 7.! So DNA must unwind when it is replicating 8.! Replication of the Escherichia coli genome (a single circular DNA) starts at a specific site (ori) and is bi-directional (Cairns, 1963). Replication as a Process 1. Double-stranded DNA must unwind.! 2. The junction of the unwound ! molecules is a replication fork.! 3. A new strand is formed by pairing ! complementary bases with the! old strand.! 4. Two molecules are made. ! Each has one new and one old ! DNA strand. ! DNA Replication is Semi-discontinuous! Continuous synthesis! Lecture 2 Outline 1.! There are many enzymes involved in DNA replication 2.! The main replicative enzyme is DNA polymerase III 3.! The enzyme is composed of several proteins 4." RNA primers are required for replication. 5.! Additional features of the replication process. Discontinuous synthesis! Arthur Kornberg (1957) Kornberg devised an in vitro assay! Protein extract from E. coli! + !template DNA! + !substrates! He guessed these would be:! dATP; dTTP; dGTP and dCTP! Set out to identify and purify ! an enzyme that could make DNA! He guessed that Mg2+ would be ! required! Discovered DNA polymerase I! Arthur Kornberg (1957) He found he could to make DNA ! in the test tube (in vitro)! Called the enzyme DNA polymerase! He purified the DNA polymerase! He guessd that ATP would be ! Needed as an energy source. Not so! Kornberg used the in vitro assay to characterize! the DNA polymerizing activity! - bases are ONLY added to the 3# end of newly ! replicating DNA! 5#! 3#! 3#! 5#! 3#! 5#! 3#! 3#! 5# Template! 5# Template! 5# Template! 3#! Found to be a single polypeptide! -therefore DNA synthesis occurs only in the! 5# to 3# direction! 928 amino acids long! DNAP I could only add bases to a primer Kornberg discovered that DNA polymerase worked! much better on single than double stranded DNA.! DNA with short single stranded regions was a good ! template.! 5#! 3#! 5#! 3#! 5#! 3#! 3#! 3#! 5# Not a template! 5# Good template! 3#! 5# Good template! Kornberg discovered that DNA polymerase I could ! not start a new DNA strand.! It could only extend a strand (the primer)that was ! base paired with a template.! 3#! 5#! 3#! 5#! 3#! 3#! 5# No reaction! 5# Good template! 3#! 5# Good template! THERE WAS A LARGE CONCEPTUAL PROBLEM! Proposal: the other strand is replicated ! “backwards and discontinously”! Consider one replication fork! 3#! 3#! 3#! 5#! Primer! 3#! 5#! Primer! Continuous replication! Continuous replication! 5#! 5#! Direction of unwinding Direction of unwinding 3#! How is the other strand replicated?! 3#! er! 5#! Prim 3#! Discontinuous replication! er! 5#! Prim 3#! 5#! 5#! Leading and lagging strands! 3#! Leading strand! 3#! 5#! Primer! Continuous replication! 5#! Evidence for the Semi-Discontinuous replication ! model was provided by Okazaki (1968)! Direction of unwinding er! 3#! 5#! Prim 3#! Discontinuous replication! er! 5#! Prim 3#! Lagging strand! 5#! Evidence for Semi-Discontinuous Replication! Evidence for Semi-Discontinuous Replication! Pulse-chase experiment ! Pulse-chase experiment ! Bacterial! culture! Bacteria are! replicating! Time zero. ! Add 3H Thymidine (T)! For a SHORT time! (i.e. seconds)! The pulse! Flood with ! non-radioactive T! Allow replication! to continue ! The chase! Purify DNA at different times Denature and measure size of all radioactive material Pulse with 3H Thymidine! A few seconds! DNA is radioactive! Radioactivity will only! be in the DNA that was ! made during the pulse! Flood the culture with non-radioactive T! Replication continues! Harvest the bacteria! at different times! Purify the DNA! Separate the strands! (using alkali conditions)! Centrifuge the single stranded DNA! Evidence for Semi-Discontinuous Replication! Pulse-chase experiment ! Results of pulse-chase experiment: after the pulse ! 3’ 5’ Chase! Leading strand Centrifuge tube Large molecule Contains aqueous solution Layer the single stranded DNA sample on top Centrifuge Pulse 5’ 3’ Small molecules Pierce the tube on the bottom Collect drops from the tube Measure the radioactivity in each drop 5’ Lagging strand Plot radioactivity per drop Evidence for Semi-Discontinuous Replication! Pulse-chase experiment! Results of pulse-chase experiment: after the chase ! 3’ 5’ Chase! Leading strand See small and large DNA just after the pulse ! Large molecule DNA purified just after the pulse Small Shows some very large molecules the leading strand 5’ Pulse and chase 3’ Large molecule And some very small ones Large the fragments from the lagging strand Evidence for Semi-Discontinuous Replication! Pulse-chase experiment! See only large DNA after a long chase ! Lagging strand 5’ DNA replication is semi-discontinuous Continuous synthesis! DNA purified just after long chase Shows only very large molecules the leading strand the fragments from the lagging strand have been joined together Discontinuous synthesis! Features of DNA Replication The enzymology of DNA polymerase I •! DNA replication is semiconservative –! Each strand of template DNA is being copied. •! DNA replication is bidirectional –! Bidirectional replication involves two replication forks, which move in opposite directions •! DNA Polymerase I has THREE different enzymatic activities in a single polypeptide •! the 5’ to 3’ DNA polymerizing activity •! DNA replication is semidiscontinuous –! The leading strand copies continuously –! The lagging strand copies in segments (Okazaki fragments) which must be joined •! a 3’ to 5’ exonuclease activity •! a 5’ to 3’ exonuclease activity DNA SYNTHESIS REACTION The 5’ to 3’ DNA polymerizing activity P 5’ 3’ P CH2 Base O CH2 P 5’ The hydrolysis of the ! phosphodiester bond! energises the reaction.! Nucleotides are added at the 3'-end of the new strand Base O P CH2 3’ 5' end of strand O CH2 Base Base O 3' P Synthesis reaction P 5' CH2 3' P P OH P products H 20 + O CH2 O P Base Base OH 3' end of strand OH Proof reading activity of the 3’ to 5’ exonuclease. Why the exonuclease activities? •! The 3'-5' exonuclease activity serves a proofreading function •! It removes incorrectly matched bases, so that the polymerase can try again. DNAP I stalls if the incorrect base is added - it cannot add the next base in the chain Proof reading activity is slow compared to polymerizing activity, but the stalling of DNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizing activity and remove the incorrect base. DNA Replication is accurate (In E. coli: 1 error/109 -1010 bases added) Why the 5’-3’ exonuclease activity? What ensures that it is so accurate?" 1) Base-pairing specificity at the active site" -!correct geometry in the active site occurs only with correctly paired bases BUT the wrong base still gets inserted 1/ 104 -105 bases added" •! The 5’-3' exonuclease activity is used to excise RNA primers in a reaction called “nick translation” 2) Proofreading activity by 3#-5# exonuclease" - removes mispaired bases from 3# end of DNA" -!increases the accuracy of replication 102 -103 fold" •! Describe the role of this later 3) Mismatch repair system" - corrects mismatches AFTER DNA replication" Is DNA Polymerase I the principal replication enzyme? In 1969 John Cairns and Paula deLucia isolated a mutant E. coli strain with only 1% DNAP I activity (polA) Other clues…. -! DNAP I is slow (600 bases added/minute – would take 100 hrs to replicate genome instead of 40 minutes) - mutant was super sensitive to UV radiation - but otherwise the mutant was fine i.e. it could divide, so obviously it could replicate its DNA - DNAP I is only moderately processive (processivity refers to the number of bases added to a growing DNA chain before the enzyme dissociates from the template) Inference: •! DNAP I may NOT BE the principal replication enzyme in E. coli Inference: •! There might be additional DNA polymerases. •! Sought other polymerases in the polA mutant So if it is not the chief replication enzyme then what does DNAP I do? The DNA Polymerase Family A total of 5 different DNAPs have been discovered in E. coli -! functions in multiple processes that require only short lengths of DNA synthesis -! has a major role in DNA repair (CairnsdeLucia mutant was UV-sensitive) -! its role in DNA replication is to remove primers and fill in the gaps left behind - for this it needs the nick-translation activity •! •! •! •! •! DNAP I: functions in repair and replication DNAP II: functions in DNA repair (proven in 1999) DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in 1999) DNAP V: functions in DNA repair (discovered in 1999) DNA SYNTHESIS REACTION DNA Polymerase III P P CH2 The "real" replicative polymerase in E. coli Base O 5' end of strand CH2 Base O •! It is fast: up to 1,000 bases added/sec/enzyme P P CH2 •! It is highly processive: >500,000 bases added before dissociating CH2 Base O Synthesis reaction P 5' CH2 P P OH P products H 20 + 3' P •! It is accurate: makes 1 error in 107 bases added; with proofreading, this gives a final error rate of 1 in 1010 overall. O Base O CH2 O P Base Base 3' OH 3' end of strand OH DNA must be “Primed” before DNA Polymerase can replicate The subunits of E. coli DNA polymerase III! Subunit! Function! Holoenzyme! Core! Enzyme! dimer! !" #" $" %" &" '" (" (#" )" *" DNA polymerase cannot initiate polymerisation 5# to 3# polymerizing activity! 3# to 5# exonuclease activity! ! and # assembly (scaffold)! Assembly of holoenzyme on DNA! Sliding clamp = processivity factor! Clamp-loading complex! Clamp-loading complex! Clamp-loading complex! Clamp-loading complex! Clamp-loading complex! de novo on double stranded DNA Okazaki and colleagues provided evidence for short stretches of RNA linked to nascent chains of DNA during replication. These RNA segments are called “primers” 1.! Sugino et al., (1972) isolated Okazaki fragments from E. coli after pulsing with 3H-U (incorporates into RNA and not DNA) Conclusions of this and later work and found it associated with newly replicated DNA. http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=426820&blobtype=pdf 2. In follow up experiments Sugino et al., (1973) isolated Okazaki fragments after a short pulse (3H-dT) by banding on a CsCl gradient. 3 4! Treatment of the Okazaki fragments with alkali 1.! There is a covalent linkage between ribonucleotides and deoxyribonucleotides in the newly synthesised DNA. 2.! RNA fragments (10 to 20 nt) are located at the 5’ end of the nascent fragments and are required for priming de novo DNA synthesis. (hydrolyses RNA but not DNA) or ribonuclease 5.! These fragments are made by a special RNA polymerase called RNA primase - this is resistant to the drug rifampicin resulted in a small reduction in density. 4! If you chop an RNA primer off the end of an Okazaki fragment you expect the density of the fragment to be reduced because RNA is denser than DNA. The initiation of the leading strand is carried out by the main E. coli RNA polymerase (which is sensitive to rifampicin) at the origin of replication, called oriC in E. coli - see below. 1 X Puzzles How do the Okazaki strands become linked to each other? Do the RNA primers stay in the new DNA? 2 3 If not how are they removed? 4 1! DNA pol III making new Okazaki DNA (red) approaches previous RNA primer (green) at X DNA pol I takes over, extending new DNA (blue) and digesting RNA primer (green) - this is called “nick translation”. DNA pol I dissociates and DNA ligase seals the nick. 2! 3 Proteins Involved in DNA Replication in E. coli 1 X 2 3 4 1! 2! 3 DNA pol III making new Okazaki DNA (red) approaches previous RNA primer (green) at X DNA pol I takes over, extending new DNA (blue) and digesting RNA primer (green) - this is called “nick translation”. DNA pol I dissociates and DNA ligase seals the nick. Protein Name Function DNA Gyrase (Topoisomerase) SSB DnaA HU PriA PriB PriC DnaB DnaC DnaT Primase DNAP III holoenzyme DNAP I Ligase Tus Unwinding DNA Single-stranded DNA binding Initiation factor Histone-like (DNA binding and bending) Primosome assembly Primosome assembly Primosome assembly DNA unwinding (helicase) DnaB chaperone Assists DnaC in delivery of DnaB Synthesis of an RNA primer Elongation (DNA synthesis) Excises RNA primer, fills in with DNA Covalently links Okazaki fragments Termination Replication - role of helicase Replication - role of DNA pol III Overall direction of replication Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ Helicase: this unwinds DNA DNA pol III adds DNA nucleotides ! to the RNA primer.! 3’ 5’ 5’ 3’ 5’ DNA pol III adds DNA nucleotides ! to the RNA primer.! DNA polymerase proofreads bases added and ! replaces incorrect nucleotides.! Replication: leading and lagging strands Overall direction of replication Replication: leading and lagging strands 2 Overall direction of replication 3’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ Okazaki fragment! 5’ 3’ 3’ 5’ 3’ 5’ 5’ Leading strand synthesis continues in a ! 5# to 3# direction.! Leading strand synthesis continues in a ! 5# to 3# direction.! Discontinuous synthesis produces a series of! 5# to 3# DNA segments - the Okazaki fragments. ! Replication: leading and lagging strands 3 Overall direction of replication Replication: leading and lagging strands 4 3’ 3’ 3’ 5’ 5’ 3’ 5’ Okazaki fragment! 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’5’ 3’ 5’ Leading strand synthesis continues in a ! 5# to 3# direction.! Leading strand synthesis continues in a ! 5# to 3# direction.! Discontinuous synthesis produces 5# to 3# DNA ! segments called Okazaki fragments. ! Discontinuous synthesis produces 5# to 3# DNA ! segments called Okazaki fragments. ! Replication: leading and lagging strands 5 1 X 3’ 5’ 3’ 2 5’ 3’ 5’ 3’5’ 3’5’ 3’ 3 5’ Leading strand synthesis continues in a ! 5# to 3# direction.! Discontinuous synthesis produces 5# to 3# DNA ! segments called Okazaki fragments. ! 4 1! 2! 3 DNA pol III making new Okazaki DNA (red) approaches previous RNA primer (green) at X DNA pol I takes over, extending new DNA (blue) and digesting RNA primer (green) - this is called “nick translation”. DNA pol I dissociates and DNA ligase seals the nick. DNA Synthesis DNA REPLICATION -!problem of going in two directions at the same time Simultaneous replication occurs via looping of lagging strand 3 Pol III synthesises leading strand 1 Helicase opens helix 2 Topoisomerase nicks DNA to relieve tension from unwinding 4 Primase synthesises RNA primer 5 6 Pol I excises RNA primer; fills gap 7 Pol III elongates primer; produces Okazaki fragment DNA ligase links Okazaki fragments to form continuous strand Pol I takes over from Pol III and runs into RNA primer Simultaneous Replication Occurs via Looping of the Lagging Strand Initiation of DNA replication, but not continuation, was shown to be sensitive to rifampicin (an antibiotic that inhibits E. coli RNA polymerase). How do we explain this? The key idea is that RNA polymerase starts the whole process of DNA replication at the origin of replication •Helicase unwinds helix •SSBPs prevent closure •DNA gyrase reduces tension •Core polymerase binds template •DNA synthesis •Not shown: pol I, ligase Initiation of the replication of the bacterial chromosome (called ORI) Initiation of the replication of the Bacterial Chromosome Initiation site is called oriC BIDIRECTIONAL REPLICATION Origin 5’ 3’ 5’ 5’ 3’ oriC 3’ RNA polymerase (RNAP) is specifically involved in starting DNA synthesis at oriC. ori ter RNAP initiates RNA synthesis within oriC. This opens up the double helix at oriC. RNAP is sensitive to rifampicin. All the primers for the both the leading strand and the Okazaki fragments, needed later, are initiated by another RNA polymerase. It is called RNA primase - it is resistant to rifampicin. 3’ 5’ Initiation of the replication of the Bacterial Chromosome Initiation site is called oriC oriC 5’ 3’ 5’ 3’ RNA polymerase starts to make RNA from two points one on each strand, going in opposite directions from inside oriC. Initiation of the replication of the Bacterial Chromosome Initiation and Termination of the replication of the Bacterial Chromosome BIDIRECTIONAL REPLICATION oriC Origin 3’ 5’ leading DNA RNA RNA 3’ DNA leading 3’ 5’ 5’ 3’ 5’ 1! After oriC is opened by RNAP, RNA primase starts to make RNA from two points one on each strand, going in opposite directions from inside oriC. 2 These are extended by DNA pol III as leading strands. 3 Okazaki fragments are made on the opposite strands. ter Replication Termination of the Bacterial Chromosome Procaryotic (Bacterial) Cairns Chromosome Replication •! Termination: meeting of two replication forks and the completion of daughter chromosomes ori •! Region 180o from ori contains replication fork traps: ori ter Bidirectional Replication Produces a Theta Intermediate ori Replication Forks Chromosome Ter sites Replication Termination of the Bacterial Chromosome One set of Ter sites arrest DNA forks progressing in the clockwise direction, a second set arrests forks in the counterclockwise direction Chromosome TerB TerA Summary •! Some of the DNA replication proteins: DNA PolIII DNA PolI DNA Ligase Primase (DnaG) Helicase (DnaB) SSB DNA gyrase (topoisomerase) •! Replication termination Replication fork traps opposite oriC Ter sites
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