The Central Dogma. Replication as a Process. DNA Replication is Semi-discontinuous!

Transcription

1 The Central Dogma DNA structure and DNA replication DNA replication (continued) RNA Synthesis rotein synthesis rof. David McConnell Smurfit Institute of Genetics DNA an emblem of the 20 th 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. 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: to and to 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 rocess 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 utline 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!

2 Arthur Kornberg (1957) Kornberg devised an in vitro assay! rotein extract from E. coli! +!template DNA! +!substrates! Set out to identify and purify! an enzyme that could make DNA! Discovered DNA polymerase I! He guessed these would be:! dat; dtt; dgt and dct! He guessed that Mg 2+ would be! required! He guessd that AT would be! Needed as an energy source. Not so! 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! Kornberg used the in vitro assay to characterize! the DNA polymerizing activity! - bases are NLY added to the 3# end of newly! replicating DNA! 5# Template! 5# Template! 5# Template! Found to be a single polypeptide! 928 amino acids long! -therefore DNA synthesis occurs only in the! 5# to 3# direction! DNA 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.! 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.! 5# Not a template! 5# Good template! 5# Good template! 5# No reaction! 5# Good template! 5# Good template!

3 THERE WAS A LARGE CNCETUAL RBLEM! Consider one replication fork! rimer! roposal: the other strand is replicated! backwards and discontinously! rimer! Direction of unwinding Continuous replication! How is the other strand replicated?! Direction of unwinding rimer! Continuous replication! Discontinuous replication! rimer! Leading and lagging strands! Leading strand! Direction of unwinding rimer! Lagging strand! rimer! Continuous replication! Discontinuous replication! rimer! Evidence for the Semi-Discontinuous replication! model was provided by kazaki (1968)! Evidence for Semi-Discontinuous Replication! ulse-chase experiment! Evidence for Semi-Discontinuous Replication! ulse-chase experiment! ulse with 3 H Thymidine! A few seconds! DNA is radioactive! Bacterial! culture! Time zero.! Add 3 H Thymidine (T)! Flood with! non-radioactive T! 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! Bacteria are! replicating! For a SHRT time! (i.e. seconds)! The pulse! Allow replication! to continue! The chase! urify the DNA! Separate the strands! (using alkali conditions)! Centrifuge the single stranded DNA! urify DNA at different times Denature and measure size of all radioactive material

4 Evidence for Semi-Discontinuous Replication! ulse-chase experiment! Centrifuge tube Contains aqueous solution Results of pulse-chase experiment: after the pulse! Chase! Leading strand Large molecule Layer the single stranded DNA sample on top ulse Centrifuge ierce the tube on the bottom Small molecules Collect drops from the tube Measure the radioactivity in each drop lot radioactivity per drop Lagging strand Evidence for Semi-Discontinuous Replication! ulse-chase experiment! See small and large DNA just after the pulse! Results of pulse-chase experiment: after the chase! Chase! Leading strand Large molecule Small Large DNA purified just after the pulse Shows some very large molecules the leading strand And some very small ones the fragments from the lagging strand ulse and chase Large molecule Lagging strand Evidence for Semi-Discontinuous Replication! ulse-chase experiment! See only large DNA after a long chase! 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!

5 Features of DNA Replication! 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 replication is semidiscontinuous! The leading strand copies continuously! The lagging strand copies in segments (kazaki fragments) which must be joined The enzymology of DNA polymerase I! DNA olymerase I has THREE different enzymatic activities in a single polypeptide! the to DNA polymerizing activity! a to exonuclease activity! a to exonuclease activity The to DNA polymerizing activity DNA SYNTHESIS REACTIN 5' end of strand products The hydrolysis of the! phosphodiester bond! energises the reaction.! 3' H 5' Synthesis reaction H Nucleotides are added at the 3'-end of the new strand 3' H H 3' end of strand 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. roof reading activity of the to exonuclease. DNA I stalls if the incorrect base is added - it cannot add the next base in the chain roof reading activity is slow compared to polymerizing activity, but the stalling of DNA I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizing activity and remove the incorrect base.

6 DNA Replication is accurate (In E. coli: 1 error/ bases added) What ensures that it is so accurate?" 1) -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/ bases added" 2) roofreading activity by 3#-5# exonuclease" - removes mispaired bases from 3# end of DNA" -!increases the accuracy of replication fold" Why the - exonuclease activity?! The -3' exonuclease activity is used to excise RNA primers in a reaction called nick translation! Describe the role of this later 3) Mismatch repair system" - corrects mismatches AFTER DNA replication" Is DNA olymerase I the principal replication enzyme? In 1969 John Cairns and aula delucia isolated a mutant E. coli strain with only 1% DNA I activity (pola) - 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 Inference:! DNA I may NT BE the principal replication enzyme in E. coli ther clues. -! DNA I is slow (600 bases added/minute would take 100 hrs to replicate genome instead of 40 minutes) - DNA 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:! 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 DNA I do? -! 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 The DNA olymerase Family A total of 5 different DNAs have been discovered in E. coli! DNA I: functions in repair and replication! DNA II: functions in DNA repair (proven in 1999)! DNA III: principal DNA replication enzyme! DNA IV: functions in DNA repair (discovered in 1999)! DNA V: functions in DNA repair (discovered in 1999) - for this it needs the nick-translation activity

7 DNA olymerase III DNA SYNTHESIS REACTIN 5' end of strand The "real" replicative polymerase in E. coli! It is fast: up to 1,000 bases added/sec/enzyme! It is highly processive: >500,000 bases added before dissociating! It is accurate: makes 1 error in 10 7 bases added; with proofreading, this gives a final error rate of 1 in overall. 3' H 5' Synthesis reaction H products 3' H H 3' end of strand DNA must be rimed before DNA olymerase can replicate The subunits of E. coli DNA polymerase III! Subunit! Function! Holoenzyme! Core! Enzyme! dimer!!" #" $" %" &" '" (" (#" )" *" 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! DNA polymerase cannot initiate polymerisation de novo on double stranded DNA kazaki 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 kazaki fragments from E. coli after pulsing with 3 H-U (incorporates into RNA and not DNA) and found it associated with newly replicated DNA In follow up experiments Sugino et al., (1973) isolated kazaki fragments after a short pulse ( 3 H-dT) by banding on a CsCl gradient. 3 Treatment of the kazaki fragments with alkali (hydrolyses RNA but not DNA) or ribonuclease resulted in a small reduction in density. 4! If you chop an RNA primer off the end of an kazaki fragment you expect the density of the fragment to be reduced because RNA is denser than DNA. Conclusions of this and later work 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 end of the nascent fragments and are required for priming de novo DNA synthesis. 5.! These fragments are made by a special RNA polymerase called RNA primase - this is resistant to the drug rifampicin 4! 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.

8 uzzles How do the kazaki strands become linked to each other? Do the RNA primers stay in the new DNA? If not how are they removed? X 1! DNA pol III making new kazaki DNA (red) approaches previous RNA primer (green) at X 2! DNA pol I takes over, extending new DNA (blue) and digesting RNA primer (green) - this is called nick translation. 3 DNA pol I dissociates and DNA ligase seals the nick X 1! DNA pol III making new kazaki DNA (red) approaches previous RNA primer (green) at X 2! DNA pol I takes over, extending new DNA (blue) and digesting RNA primer (green) - this is called nick translation. 3 DNA pol I dissociates and DNA ligase seals the nick. roteins Involved in DNA Replication in E. coli rotein Name DNA Gyrase (Topoisomerase) SSB DnaA HU ria rib ric DnaB DnaC DnaT rimase DNA III holoenzyme DNA I Ligase Tus Function Unwinding DNA Single-stranded DNA binding Initiation factor Histone-like (DNA binding and bending) rimosome assembly rimosome assembly rimosome 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 kazaki fragments Termination Replication - role of helicase Replication - role of DNA pol III verall direction of replication verall direction of replication Helicase: this unwinds DNA DNA pol III adds DNA nucleotides! to the RNA primer.! DNA pol III adds DNA nucleotides! to the RNA primer.! DNA polymerase proofreads bases added and! replaces incorrect nucleotides.!

9 Replication: leading and lagging strands Replication: leading and lagging strands 2 verall direction of replication verall direction of replication kazaki fragment! 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 kazaki fragments.! Replication: leading and lagging strands 3 Replication: leading and lagging strands 4 verall direction of replication kazaki fragment! Leading strand synthesis continues in a! 5# to 3# direction.! Discontinuous synthesis produces 5# to 3# DNA! segments called kazaki fragments.! Leading strand synthesis continues in a! 5# to 3# direction.! Discontinuous synthesis produces 5# to 3# DNA! segments called kazaki fragments.! Replication: leading and lagging strands X 3 Leading strand synthesis continues in a! 5# to 3# direction.! Discontinuous synthesis produces 5# to 3# DNA! segments called kazaki fragments.! 4 1! DNA pol III making new kazaki DNA (red) approaches previous RNA primer (green) at X 2! DNA pol I takes over, extending new DNA (blue) and digesting RNA primer (green) - this is called nick translation. 3 DNA pol I dissociates and DNA ligase seals the nick.

10 DNA RELICATIN DNA Synthesis -!problem of going in two directions at the same time Simultaneous replication occurs via looping of lagging strand 2 Topoisomerase nicks DNA to relieve tension from unwinding 3 ol III synthesises leading strand 1 Helicase opens helix 4 rimase synthesises RNA primer 5 ol I excises RNA primer; fills gap 6 7 ol III elongates primer; produces kazaki fragment ol I takes over from ol III and runs into RNA primer DNA ligase links kazaki fragments to form continuous strand Simultaneous Replication ccurs 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? Helicase unwinds helix SSBs prevent closure DNA gyrase reduces tension Core polymerase binds template DNA synthesis Not shown: pol I, ligase The key idea is that RNA polymerase starts the whole process of DNA replication at the origin of replication (called RI) Initiation of the replication of the bacterial chromosome Initiation of the replication of the Bacterial Chromosome BIDIRECTINAL RELICATIN Initiation site is called oric rigin oric ori ter RNA polymerase (RNA) is specifically involved in starting DNA synthesis at oric. RNA initiates RNA synthesis within oric. This opens up the double helix at oric. RNA is sensitive to rifampicin. All the primers for the both the leading strand and the kazaki fragments, needed later, are initiated by another RNA polymerase. It is called RNA primase - it is resistant to rifampicin.

11 Initiation of the replication of the Bacterial Chromosome Initiation site is called oric oric 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 oric leading RNA DNA RNA DNA leading 1! After oric is opened by RNA, 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 kazaki fragments are made on the opposite strands. BIDIRECTINAL RELICATIN ori ter rigin rocaryotic (Bacterial) Cairns Chromosome Replication ori ter Bidirectional Replication roduces a Theta Intermediate Replication Forks Replication Termination of the Bacterial Chromosome! Termination: meeting of two replication forks and the completion of daughter chromosomes! Region 180 o from ori contains replication fork traps: ori Chromosome Ter sites

12 Replication Termination of the Bacterial Chromosome ne set of Ter sites arrest DNA forks progressing in the clockwise direction, a second set arrests forks in the counterclockwise direction Chromosome Summary! Some of the DNA replication proteins: DNA oliii DNA oli DNA Ligase rimase (DnaG) Helicase (DnaB) SSB DNA gyrase (topoisomerase) TerB TerA! Replication termination Replication fork traps opposite oric Ter sites