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Suggested problems: 1-3, 5, 7, 9-11, 13, 14, 18, 19, 21-24 see pp 350-351. Omit (now) recombination, pp 343-346.
Watson-Crick Watson-Crick model of model for Semiconservative Semiconservative DNA DNA replication replication
How does a double helix produce another double helix with precision?
5’ “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” (Watson & Crick, 1953). 3’ 5’ 3’ 5’ 3’
Three Hypotheses to explain DNA replication Supported by experiment
Semiconservative DNA replication
The two strands in the double helix separate, and then each strand serves as template for the synthesis of a new (complementary) strand. After replication has been completed, each of the two duplexes has one old and one newly synthesized strand. Conservative and dispersive modes of replication do not make much sense, and are not supported by experiments.
Isolate DNA from E. and Hughes experiment in 1958. one DNA replication). both chromatids of each chromosome were labeled. 12. heavy and light dsDNA molecules move to different positions in a density gradient and can be separated from each other. isolate DNA and subject to ultracentrifugation.3 p324-3255 in your text) The Key Experiment… Grow a culture in 15NH4Cl and then transfer to 14NH4Cl. How does this show semiconservative replication? See next slide heavy mixed light 2 . Woods. Allow cells to grow one generation (i. Subject DNA to ultracentrifugation in a density gradient of CsCl solution. treated the roots with Colchicin.Evidence for Semiconservative Replication (See Fig. They labeled DNA with 3H-T. Thus. The results (below) support semiconservative replication Eukaryotic DNA replication is also semiconservative Eukaryotic DNA also replicates semiconservatively as shown in Fava bean by the Taylor. providing evidence for semiconservative replication. fixed and prepared for microscopy. Migration to an intermediate position suggests the presence of one heavy (15N) and one light (14N) chain in dsDNA. Result: Semidense (15N/ 14N) DNA migrates to an intermediate position.2-12. after labeling at interphase. whereas at the second metaphase only one chromatid was labeled.e. At the first metaphase.. coli cultures grown in 14NH4Cl (light) and 15NH 4 Cl (heavy). Result: Heavy (dense) DNA migrates faster than light DNA towards the bottom of centrifuge tube.
10. Enzymes (DNA polymerases) can only add a nucleotide to a free OH group at the 3'-end of a growing chain (Fig. Starts at specific sites (origins) and moves in opposite directions using two replication “forks”. 12. light blue) DNA chain Other features of DNA replication • Bidirectional.7-8. A replication Fork Each fork at a replicon has a lagging strand and a leading strand 3 . • Semi-discontinuous. 12. p329-330). One strand (leading) replicates continuously and the other (lagging) discontinuously (Fig. p331). • In the 5’ .3’ direction.because they each have a newly synthesized (labeled.
12. 4 . and other protein factors) Other aspects of replication : DNA replication begins at a specific site or sites called origin. theta scheme. Prokaryotes.4. 12. at a rate of 1. (Fig. Circular DNA. multiple (500-25. dTTP) Enzymes (helicase. Viruses. rolling circle. ligase. p327). 12. one origin. polymerases.000 nt per second (20-40 min) – (Fig. one origin. dCTP.6. Linear DNA. p326). p328). The origin is recognized by specific initiator proteins. topoisomerase.000) origins at a rate of 10-50 nt per second (3-4 min to hours)— (Fig. Circular DNA. Eukaryotes.5.One enzyme dimer synthesizes both the leading and the lagging strand DNA replication requires: • • • • Template (ssDNA) Primer (RNA) Substrates (dNTPs: dATP. primase. dGTP. unidirectional (one fork).
Replication of prokaryotic DNA --Theta model Replicon (Unidirectional Replication) Replication of linear DNA using multiple origins 5 .
*Bloom syndrome is due to a mutant helicase. Pol α in eukaryotes) When all of the above factors are in place. and the forks move in opposite direction Initiation of Replication at an origin Left Fork Right Fork 6 . Each origin produces a replicon with two forks. DNA polymerase can bind to the template and start synthesis. Initiation and unwinding • Binding of initiator proteins to the origin and strand separation (melting) by helicase* • ssDNA strand stabilization by ssDNA-binding proteins • Torsional strain release by gyrase or topoisomerase • Priming (primase: a special RNA polymerase.Multiple origins of replication in eukaryotic DNA Steps in DNA Replication 1.
RNA Primer is extended with DNA DNA fragments (Okazaki fragments) are spliced (ligated) RNA Primer is removed and replaced with DNA 7 . 5’-3’ exonuclease) • Splicing of fragments (DNA ligase links the Okazaki fragments after Pol I fills the gap) 5’ to 3’ chain elongation by adding new nucleotides to the 3’-end of the growing chain 3. Termination occurs when two forks meet each other or a specific termination signal is encountered. 3’-5’ exonuclease ) • Excision of RNA primer + gap filling (Polymerase I removes RNA nucleotide and replaces it with DNA nucleotide.Steps in DNA Replication. the chain grows in the 5’-3’ direction) • Proofreading (DNA polymerase III. cont. Elongation • Addition of dNTPs (chain elongation by DNA pol III starting at the 3’-end of the RNA primer. 2. Thus.
many replication origins are needed.) Not surprising. ε. Mismatch repair corrects a wrong base in newly replicated DNA. Organelle DNA is synthesized by γ. δ. 8 . Cancer cells do not lose telomerase activity. In eukaryotes. After replication. The enzyme has a fixed position within the nucleus. proofreading activity. The telomeres or the ends of eukaryotic chromosomes are synthesized by a special reverse transcriptase (telomerase). the nucleosome reassembles. α. the nucleosome must dissociate (histones removed) to expose the naked DNA to the replication machinery. DNA replication has extremely high as opposed to 4 (α. Lack of telomerase activity leads to shortening of chromosome ends and is implicated in aging and eventually death. There are other DNA polymerases. fidelity as a result DNA polymerase’s accuracy. δ. The same enzyme molecule (a dimer) synthesizes both the leading and the lagging strand. which requires doubling the amount of histones. they are involved in DNA repair. The newly replicated DNA is recognized based on its lack of methylation Additional Points There are 2 DNA polymerases (I and III) in prokaryotes Additional Points (cont. γ) in eukaryotes. and they are used only once under the control of licensing proteins (binding of licensing proteins). ε. the DNA template is threaded through it. and post-replication mismatch repair.A misincorporated base (A in this example) is removed and then replaced with a correct base (C in this example). What would be the result without licensing? Prior to replication. Okazaki fragments are synthesized by Pol III.
serves as a primer. and its 3’end is extended by DNA polymerase 9 .has a gap Telomerase brings an RNA template and ﬁlls the gap This gap cannot be ﬁlled by DNA polymerases because they cannot extend a chain in the 3’-5’ direction Further extension of the chromosome end by telomerase The chain extended by telomerase loops back.
3’ direction (Pol I). Exonucleases – 5’ . • 1. Specific (Restriction endonucleases) 10 . DNA-dependent RNA polymerases • – Require template but not primer. Pol α. • 2. Endonucleases Cleaves phosphodiester bonds internally. Non-specific (DNases. • – Synthesize RNA in the 5’ .5’ (Pol III). • – Extend primer in the 5’ .5’ exonucleases: digest in the 3’ . RNases). – 3’ .3’ direction. • – Example: Pol III.DNA Synthesis and Hydrolysis Enzymes DNA-dependent DNA polymerases • – Require template and primer.3’ direction.3’ exonucleases: digest DNA or RNA in the 5’ .
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