Semiconservative DNA replication. Meselson and Stahl

Transcription

1 DNA replication

2 Semiconservative DNA replication Meselson and Stahl

3 Hartl Replication of DNA

4 New nucleotides are added to DNA only during replication in the 5-3 direction

5 How double helix unwind

6 DNA synthesis takes place simultaneously but in opposite directions on the two DNA templates ss ss Direction of synthesis: 5-3

7 DNA synthesis is continuous on one template of DNA and discontinuous on the other Lagging strand replication requires the formation of Okazaki fragments

8 Priming of DNA synthesis with a RNA segment (RNA primer) nts long (3 -OH) Primase synthesizes short streches of RNA nucleotides, provinding a 3 -OH group to which DNA polymerase can add DNA nucleotides

9 On the leading strand, where replication is continuous, aprimerisrequiredonly at the 5 end of the newly synthesized strand On the lagging strand, with discontinuous replication, a new primer must be generated at the begining of each Okazaki fragment

10 Helicase, Primase, SSB and DNA polimerase DNA helicase unwinds DNA by binding to the lagging-strand template at each replication fork and moving in the 5-3 direction along the strand by breaking hydrogen bonds Primase forms a complex with helicase (primosome) SSB (single-stranded binding) proteins stabilize the exposed single-stranded DNA

11 DNA polimerases in E. coli DNA polimerase III DNA polimerase I

12 5-3 polymerase activity DNA polymerase III

13 Proofreading role of DNA polymerase III Hydrolysis due to 3-5 exonucleolytic activity 3-5 exonuclease activity

14 DNA polymerase III- a large multiprotein complex α- actividade polimerase 5-3 ε- exonuclease 3-5 β- aumenta processividade da enzima θ- necessária ao assembly de α ε τ τ- mantém estrutura do dímero e contacta com DnaB (primase) Modelo assimétrico da DNA polimerase III apoia o modelo da replicação simultânea das duas cadeias

15 A β subunit tethers the core of E. coli DNA polymerase III to DNA thereby increasing its processivity

16 DNA polymerase I st ribonucleotide of RNA primer is trifosphatated 5-3 exonuclease activity 5-3 polymerase activity dntp DNA ligase 5 3 Nick 3 5 DNA polymerase I also have 3-5 exonuclease activity

17 ACTIVITIES DNA polymerase I DNA polymerase III Synthesis DNA polymerase I DNA polymerase III Removes incorrect nucleotide DNA polymerase I Displaces incorporated nucleotides

18 Characteristics of DNA polymerases in E. coli

19 Klenow fragment (large DNA polymerase I fragment) 5-3 polymerase activity DNA polymerase I (103 kda) 5-3 exonuclease activity 3-5 exonuclease activity DNA polymerase I has the unique ability to start replication in vitro at a nick in DNA 5-3 polymerase activity Klenow fragment (large DNA polymerase I fragment) (68 kda) 5-3 exonuclease activity 3-5 exonuclease activity N proofreading synthesis COOH Small fragment (35 kda)- 5-3 exonuclease activity

20 Topoisomerase I

21 Replisome

22 Cooper 4.19 Origin of replication

23 Model of initiation of replication at E. coli oric (Konberg and collab.) 245 bp Ligation of DnaA (initiator proteins) occurs when DNA is negatively supercoiled DNA forced to unwind in 13-mers Activates DnaG (primase) Typical 13-mer Typical 9-mer GATCTATTTATTT TTATCCACA Unwinding allows helicase and other SSB proteins to attach to single-stranded DNA

24 Relationship between E. coli replication proteins at a growing fork In this model, DNA must form a loop so that both strands can replicate simultaneously

25 DNA polymerase III- a large multiprotein complex α- actividade polimerase 5-3 ε- exonuclease 3-5 β- aumenta processividade da enzima θ- necessária ao assembly de α ε τ τ- mantém estrutura do dímero e contacta com DnaB (primase) Modelo assimétrico da DNA polimerase III apoia o modelo da replicação simultânea das duas cadeias

26 Model of DNA replication in E. coli, where two units of DNA polymerse III are connected The lagging strand loops around so that 5-3 synthesis can take place on both antiparallel strands

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28 Unidirectional vs bidirectional replication Circular DNA molecule Linear DNA molecule

29 Modes of Replication Theta Rolling circle Linear

30 Theta replication is a type of common in E. coli and other organisms possessing circular DNA Producing single-stranded templates for the synthesis of new DNA. A replication buble forms, usuallly having a replication fork at each end (bidirectional replication) The fork proceeds around the circle Two DNA molecules are produced Double-stranded DNA unwinds at the replication of origin The products of theta replication are two circular DNA molecules

31 Rolling-circle replication (circular DNA) Nick Displaced strand 5 Cleavage in one of the nucleotide strands 3 -OH at the nick is the growing point where DNA synthesis begins. The inner strand is used as a template Takes place in some virus and in the F factor of E. coli

32 The 3 end grows around the circle giving rise to the name rollingcircle model 3 Cleavage may release a single-stranded linear DNA and a double-stranded circular DNA or The cycle may be repeated

33 The cycle may be repeated Two copies (or more) of same sequence of linear DNA

34 The linear molecule circularizes after serving as a template for the synthesis of a complementary strand 2nd revolution New synthesized DNA (discontinuous replicationlagging strand) 1st revolution Or either before serving as a template

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36 - D-loop - One or several linear molecules

37 Termination of DNA replication in E. coli Replication termini in E. coli are located beyond the point at which the replication forks actually meet One of the replication forks has proceeded some distance past the halfway point. This does not happen during DNA replication in E.coli because of the action of the Tus proteins

38 The role of terminator sequences during DNA replication in E. coli β-strands facing the fork

39 Eucaryotic replication

40 Linear DNA replication takes place in eukaryotic chromosomes Replication begins and is bidireccional Synthesis starts at all the origins of replication Replication bubles fuse where they meet

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42 Structure of yeast origin of replication ARS- autonomously replicating sequence, that acts as na origin of replication in S. cerevisae < 200 bp A, B1, B2 and B3- functional sequences Melting of the helix occurs within the subdomain B2, induced by the attachment of ARS binding protein (ABFI) to subdomain B3. The proteins of the origin of replication complex (ORC) are permanently attached to subdomains A and BI

43 Number and lenght of replicons Organism Number of replication origins Average lenght of replicon (bp) Escherichia coli (bacterium) Saccharomyces cerevisae (yeast) Drosophila melanogaster (fruit fly) Xenopus laevis Mus musculus (mouse)

44 Events in DNA replication in E. coli and eukaryotes

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46 Priming of DNA synthesis In eukaryotes the primase forms a complex with DNA polymerase α, which is shown synthesizing the RNA primer followed by the first few nucleotides of DNA

47 Removal of the RNA primer from each Okazaki fragment in eukaryotes, by FEN I endonuclease There appears to be no DNA polymerase with 5-3 exonuclease activity in eukaryotes The flap endonuclease FEN I cannot initiate primer degradation because its activity is blocked by the triphosphate group present at the 5 end of the primer

48 Two models for completion of lagging strand replication in eukaryotes

49 Telomer replication

50 Sequências de DNA nas extremidades dos cromossomas The telomere has a protruding end with a G-rich repeated sequence Como se resolve o problema da extremidade 3 projectada originada durante o processo de replicação do DNA?

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52 The mechanism of restoring the ends of a DNA molecule in a chromosome relies on an enzyme called TELOMERASE 3 5 Telomerase elongates the template DNA strand at the 3 end 5 3 The telomerase contains na internal RNA with a sequence complementary to the telomere repeat

53 Mechanism of action of telomerase

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55 G-quartet structure formed by hydrogen bonding between four guanine bases present in a single DNA strand folded back upon itself

56 Models of telomere structure in Oxytricha and Tetrahymena

57 Replication slippage A trinucleotide repeat in the act of replication The 3 end of the growing strand momentrily detaches from the template and reanneals to the template at a point upstream from its original location Continued replication duplicates the region between the points of detachment and reannealing Mismatch repair of the shorter strand creates a duplex with a trinucleotide expansion Doenças neurodegenerativas (doenças de expansão de repetições de trinucleotídeos)

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