DNA Replication PDF

DNA Replication Dr Mark Carlile Dale 106 [email protected] Dr Mark Carlile : DNA Replication 1 Overview of my lectures on PHA111 All of my lectures are broadly focused on molecular biology: Wk 27: Nucleic acid biochemistry Wk 28: DNA replication Wk 28: Transcription Wk 28: Translation (p...

DNA Replication Dr Mark Carlile Dale 106 [email protected] Dr Mark Carlile : DNA Replication 1 Overview of my lectures on PHA111 All of my lectures are broadly focused on molecular biology: Wk 27: Nucleic acid biochemistry Wk 28: DNA replication Wk 28: Transcription Wk 28: Translation (protein synthesis) Wk 27: Protein folding and sorting Wk 28: Biotechnology Wk 28: Recap and some biological therapeutics stuff Dr Mark Carlile | Nucleic acid biochemistry 2 Recommended textbook: Dr Mark Carlile : DNA Replication 3 Learning outcomes A knowledge of: The biochemical processes used in the replication of DNA in prokaryotes and eukaryotes The experiments that have been used to understand DNA replication How DNA replication is controlled as part of the cell cycle Dr Mark Carlile : DNA Replication 4 DNA replication – simplistic structure but complex mechanism Watson and Crick’s seminal paper describing the DNA double helix ended with the statement: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” Dr Mark Carlile : DNA Replication 5 Why is DNA a helix? Lets explore why DNA is a Helix using this online tool at the university of Loiverpool: (the place where I learned all abot DNA biochemistry!) https://www.liverpool.ac.uk/sbs/DNA_Topology/HelixL.htm The reason why is not mystical its just chemistry! Dr Mark Carlile : DNA Replication 6 Hersey Chase Experiment (1952) Alfred Hershey and Martha Chase conducted a series of experiments to prove that DNA was the genetic material Viruses (T2 bacteriophage) were grown in one of two isotopic mediums in order to radioactively label a specific viral component Viruses grown in radioactive sulphur (35S) had radiolabelled proteins (sulphur is present in proteins but not DNA) Viruses grown in radioactive phosphorus (32P) had radiolabelled DNA (phosphorus is present in DNA but not proteins) The viruses were then allowed to infect a bacterium (E. coli) and then the virus and bacteria were separated via centrifugation - The larger bacteria formed a solid pellet while the smaller viruses remained in the supernatant The bacterial pellet was found to be radioactive when infected by the 32P–viruses (DNA) but not the 35S–viruses (protein) This demonstrated that DNA, not protein, was the genetic material because DNA was transferred to the bacteria Dr Mark Carlile : DNA Replication 7 DNA Replication DNA replication is described as semiconservative a) Semiconservative replication gives two duplex DNA molecules each of which contains one old strand and one new strand. b) Conservative replication gives two duplexes, one of which contains two old strands, and a second containing two new strands c) Dispersive replication gives two daughter duplexes each of which contains strands that are a mixture of new and old strands Experiments by Meselson and Stahl (1958) proved the semiconservative replication model. Dr Mark Carlile : DNA Replication 8 Meselson and Stahl experiments (1958) A centrifuge was used to separate DNA molecules labeled with isotopes of different densities. This experiment revealed a pattern that supports the semiconservative model of DNA replication. Question: If you continued this experiment for two more generations (as Meselson and Stahl actually did), what would be the composition (in terms of low- and intermediate-density) of the fourth generation DNA? Dr Mark Carlile : DNA Replication 9 DNA Replication initiation The process of DNA replication is broadly similar in prokaryotes and eukaryotes although the process is more complicated in eukaryotic cells Replication is initiated at an ‘origin of replication’ that gives rise to two replication forks The eukaryote genome is much larger than the prokaryote genome and replication involves multiple origins Dr Mark Carlile : DNA Replication 10 DNA replication – The most basic mechanistic overview Replication Bubble A branch point in a replication eye at which DNA synthesis occurs is called a replication fork. A replication bubble may contain one or two replication forks (unidirectional or bidirectional replication) DNA replication is almost always bidirectional Prokaryotic and bacteriophage DNAs have one replication origin (point where DNA synthesis is initiated) Reniji Okazaki elucidated the semidiscontinuous model of DNA replication Dr Mark Carlile : DNA Replication 11 Replication initiation – in Prokaryotes There are 4 copies of a 9-bp sequence that bind DnaA proteins – once all binding sites are occupied they cooperatively recruit more DnaA proteins = DnaA barrel formation This helps open-up a local AT-rich region of the DNA via torsional stress A pair of replication forks are then generated Dr Mark Carlile : DNA Replication 12 Replication initiation – in Prokaryotes DnaB is recruited to the replication fork to initiate the formation of the pre-priming complex DnaB = Helicase enzyme : breaks base-pairs (hydrogen bonds) The open DNA strands are then covered with SSBs : single stranded binding proteins SSBs : stop the strands re-annealing (base-pairing) and protect the DNA from attack by free radicals and nuclease enzymes The initiation of DNA replication is now complete and the next phase : ELONGATION is started Dr Mark Carlile : DNA Replication 13 The polymerisation reaction The synthesis of the new strand is carried out by DNA-dependent DNA polymerase enzymes DNA is generated in the 5’ to 3’ direction The polymerase moves along the template strand in the 3’ to 5’ direction Dr Mark Carlile : DNA Replication 14 DNA Template Strand 3’-end of the new strand DNA Template Strand Polymerisation mechanism Electron withdrawing PP-O-P Favorable energetics Dr Mark Carlile : DNA Replication 15 Polymerisation mechanism DNA Template Strand Electron withdrawing PP-O-P Nucleophilic attack: need some electron withdrawal at the phosphate!! 5’-end of the new strand Unfavorable Energetics Dr Mark Carlile : DNA Replication 16 Prokaryotic elongation Primase enzyme (DnaG) binds near to the helicase and starts to synthesise the RNA primer on the leading strand (Primosome) Single strand binding proteins stabilize the lagging strand DNA polymerase III holoenzyme clamps to the leading strand and synthesizes DNA The DNA ploymerase III holoenzyme complex The DNA polymerase III holoenzyme is a multi-subunit complex, which consists of 17 polypeptides. It contains four subassemblies. 1) The core polymerase consists of three subunits: α (the polymerase); ε (the 3'–5' exonuclease); and θ (the stimulator of the 3'–5' exonuclease). 2) The τ subunit is responsible for dimerization of the core DNA polymerase. 3) The sliding clamp comprises two homodimers of the β subunit, which provides the ring structure that is needed for processivity. 4) Five subunits have clamp-loader functions — γ, δ, δ', χ and ψ. 5) There is also an RNA polymerase enzyme associated with the core enzyme Do not stress about remembering this Dr Mark Carlile : DNA Replication 17 Semi-discontinuous replication DNA synthesis is carried out by DNA polymerase in the 5’ to 3’ direction The Polymerase enzyme inserts the 5’ nucleotide first and extends towards the 3’ end So the template DNA molecule is always used in the 3’ to 5’ direction The lagging strand is generated via the synthesis of multiple “Okazaki Fragments” The lagging strand is generated in the opposite direction to the movement of the replication fork. Dr Mark Carlile : DNA Replication 18 Dealing with the Okazaki fragments All DNA replication is started with a short RNA primer – this allows proofreading of the newly synthesized strand The primase enzyme does this in E.coli The primer is removed by the exonuclease activity of the polymerase complex So that both strands of DNA can be replicated at the same time two DNA polymerase enzymes are tethered together – each replicating one strand One continuously: in the 5’ to 3’ direction (leading strand) One discontinuously : in the 5’ to 3’ direction on the lagging strand This is called the REPLISOME Dr Mark Carlile : DNA Replication 19 Dealing with the Okazaki fragments The lagging strand is looped over the top of the replisome so that both polymerases move in the same direction Topoisomerase enzymes proceed ahead of the replisome The Okazaki fragments are joined together by a ligase enzyme (Ligation) - in fact DNA polymerase III is replaced by another polymerase (Pol I) which removes the RNA primer on the Okazaki fragment prior to the ligation Dr Mark Carlile : DNA Replication 20 DNA replication - Elongation The replication forks can only progress a short distance without before there is a topological problem The double helix requires rotation as the helix is opened to stop over-winding ahead of the replication fork Specialised enzymes: Topoisomerases are able to alleviate these problems: Type I : introduce a break in one strand, pass the other strand through and then reseal the break Type II : break both strands and then pass a double helix through the gap before re-sealing the break The breaks in DNA are covalently attached to the enzymes – so they don’t loose the ends! Dr Mark Carlile : DNA Replication 21 Overcoming the topological problems of DNA replication Dr Mark Carlile : DNA Replication 22 Termination of the replication process In E.coli – the two replicons meet 180O away from the origin of replication A regulatory mechanism is in place to make sure that the replicon meet at a specific point – if one get there first then it will wait for the other one to arrive before signaling that DNA replication is complete Specific terminator sequences signal that the replicon is approaching the stop sequence If the replicon meets a transcription bubble (mRNA synthesis) then it will wait and not overtake Topoisomerase activity: Once the two “daughter” DNA molecules are generated they are interlinked (catenated) Unlinking is carried out by Topoisomerase enzymes Cell division: The separated DNA molecules are then segregated awaiting cell division wherein each cell will receive a DNA molecule Dr Mark Carlile : DNA Replication 23 eplicaubunit termi. Likerightof the mably, t with sliding e most highly 5¿ ex- Eukaryotic DNA replication There are a remarkable level of similarities between prokaryotic and eukaryotic DNA replication mechanisms. However: Because of the complexity of eukaryotic DNA structures (chromatin) eukaryotic replication forks are much slower than that of prokaryote replication forks Eukaryotic replication forks move at around 50 bp per second Due to the size of eukaryotic genomes multiple replication Section 30-4. Eukaryotic Replication 1205 forks are required: 50,000 – 100,000 per mammalian cell DNA Figure 30-44 ElectronReplicating micrograph of a Drosophila fragment of replicating Drosophila DNA. The arrows indicate its multiple replication eyes. [From Kreigstein, H.J. and Hogness, D.S., Proc. Natl. Acad. Sci. 71, 136 (1974).] Dr Mark Carlile : DNA Replication 24 Eukaryotic DNA replication In eukaryotes clusters of of about 20-50 replicons initiate simultaneously at defined time in S-phase based upon accessibility to initiation factors (mitogens): Early S-phase clusters: Late S-phase clusters: Centromeric and telomeric DNA Euchromatin – transcriptionally active DNA Heterochromatin – transcriptionally silent(ish) DNA (??) is replicated last In yeast: The minimum length of a DNA molecule that will support replication is 11 bp with the sequence: [A/T]TTTAT[A/G]TTT[A/T] Additional copies of this sequence are required for optimal replication efficiency This sequence - is bound by the Origin Replication Complex (ORC) whish is activated by cyclin dependent kinases (CDKs) - Facilitates/initiates the opening of the DNA duplex In mammals: Defined ORCs have not been isolated (yet) – it is believed that replication my initiate at random in areas of repetitive DNA sequence (??) Dr Mark Carlile : DNA Replication 25 Eukaryotic DNA replication Eukaryotic DNA replicons can only initiate once per cell – this is so that DNA is fully and controllably replicated prior to cell division. This limits the introduction of mutations. Origin of Replication Complex A protein licensing factor complex is required for the initiation of DNA replication and is inactivated after use and is only able to gain access to the nucleus when the nuclear envelope dissolves in mitosis Basically the origin is - Identified (binding of ORC) - Set-up (binding of initiation factors) - Checked - Initiated GO!!!!!..... Do not worry about the specifics here Dr Mark Carlile : DNA Replication 26 Eukaryotic DNA replication Replication of chromosome ends (Telomeres) The ends of the chromosomes cannot be replicated by semi-discontinuous replication because there is no DNA to elongate once the RNA primer is removed from the 5’-end of the lagging strand This could potentially lead to the loss of genetic material To overcome this problem eukaryotic chromosomes have a hundreds of copies of a non-coding repeat sequence: TTAGGG The 3’-end overhangs the 5’-end enzyme telomerase associates with short RNA molecules that are yotic The DNA replication partially complementary to this sequence The RNA acts as a template for the addition of the repeats to the 3’-end overhangs n= >200 89 The complementary strand is synthesized by normal lagging strand synthesis Fig. 3. of human telomeric DNA (n = several hundred). which leaves theSequence 3’-overhang Telomerase activity is repressed in somatic cells leading to a gradual loss of DNA and shortening of the chromosomes – May cause some of the problems in the aging process elongation (polymerization) andDr Mark translocation. The complementary strand is Carlile : DNA Replication 27 Overview What we have covered: DNA replication: Basic process in prokaryotes (and a simplified mechanism in eukaryotes) Semi-conservative replication Semi-discontinuous replication Why DNA needs to be synthesized in the 5’ to 3’ direction (energetics) Dr Mark Carlile : DNA Replication 28