BrownLect5_Revised_2009 - DNA Replication, part I...

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Unformatted text preview: DNA Replication, part I Properties of DNA replication • DNA polymerase • semi-conservative • bi-directional • semi-discontinuous • RNA primed Watson, J.D. and F.H.C. Crick. 1953. Genetical implications of the structure of deoxyribonucleic acid. Nature. 171:964-967 DNA polymerase •Kornberg, Lehman, Bessman, and Simms, 1956 It always seemed to me that a biochemist devoted to enzymes could, if persistent, reconstitute any metabolic event in the test tube as well as the cell does it. In fact better! Without the constraints under which an intact cell must operate, the biochemist can manipulate the concentrations of substrates and enzymes and arrange the medium around them to favor the reaction of his choice.--Arthur Kornberg acid soluble (nucleosides/nucleotides) ATP Mg2+ E. coli extract thymidine acid insoluble (polynucleotides) DNase acid soluble Hypothesized that existing chains were being elongated by the enzyme But... all three dNTPs had to be present for dTTP to be incorporated H H And... the newly synthesized DNA had the same AT to GC ratio as the “primer” DNA H OH OH "These results suggest that enzymatic synthesis of DNA by the "polymerase" of E. coli represents the replication of a DNA template." Lehman, I. R., Zimmerman, S. B., Adler, J., Bessman, M. J., Simms, E. S., and Kornberg, A. (1958) Proc. Natl. Acad. Sci. U. S. A. 44, 1191–1196 From the analysis of purified DNA polymerase I, several important properties became apparent: • can’t start DNA chains de novo--a primer is required • synthesizes DNA in the 5’ to 3’ direction ONLY • not highly processive or rapid • 3’ to 5’ exonuclease activity allows proof-reading DNA polymerization http://www.steve.gb.com/science/dna_replication.html Beese, L. S., Derbyshire, V., and Steitz, T. A., 1993. Science 260:352 - 355 • polymerase and 3’ to 5’ exonuclease sites are distinct • proof-reading increases fidelity from 10-4 to 10-7 DNA polymerase I is a repair enzyme • the polymerase responsible for replicating the E. coli chromosome in vivo is DNA polymerase III, discovered by Tom Kornberg • despite this, DNA pol I is the prototype for all DNA polymerases, enzymatically and structurally. pol I pol beta phage rb69 http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb3_3.html SO... • W & C propose that DNA unwinds to provide a template for it’s reproduction AND • Kornberg identifies an enzyme that synthesizes DNA using an existing DNA strand as the template What happens in vivo? Replication is semi-conservative •Meselson and Stahl, 1958 Three possibilities: Weaver, Molecular Biology, Figure 20.1 Replication is semi-conservative •Meselson and Stahl, 1958 Three possibilities: Weaver, Molecular Biology, Figure 20.1 Meselson and Stahl. The Replication of DNA in Eschericia coli. PNAS (1958) 44: 675. Weaver, Molecular Biology, Figure 20.3 Replication is bi-directional • John Cairns, 1963; Huberman and Riggs, 1968 • • • • • • label cells with 3H-thymidine lyse cells spread DNA on a slide coat with photographic emulsion wait a long time develop Cairns. The bacterial chromosome and its manner of replication as seen by autoradiography. J Mol Biol. 1963 Mar;6:208-13. Replication is bi-directional • John Cairns, 1963; Huberman and Riggs, 1968 • • • • • • label cells with 3H-thymidine lyse cells spread DNA on a slide coat with photographic emulsion wait a long time develop Weaver, Molecular Biology, Figure 20.10 Cairns. The bacterial chromosome and its manner of replication as seen by autoradiography. J Mol Biol. 1963 Mar;6:208-13. • observed what were called “theta” structures • two forks are evident--are BOTH sites of DNA replication? Replication is bi-directional • John Cairns, 1963; Huberman and Riggs, 1968 • • • • • • label cells with 3H-thymidine lyse cells spread DNA on a slide coat with photographic emulsion wait a long time develop Weaver, Molecular Biology, Figure 20.10 Cairns. The bacterial chromosome and its manner of replication as seen by autoradiography. J Mol Biol. 1963 Mar;6:208-13. • observed what were called “theta” structures • two forks are evident--are BOTH sites of DNA replication? Replication is bi-directional • John Cairns, 1963; Huberman and Riggs, 1968 Experiment: • pulse-label replicating cells with 3H-thymidine • then chase with cold thymidine • extract DNA and stretch on glass slide • autoradiograph the fibers J Mol Biol. 1968 Mar 14;32(2):327-41. Replication is bi-directional • John Cairns, 1963; Huberman and Riggs, 1968 Experiment: Predictions: • pulse-label replicating cells with 3H-thymidine • then chase with cold thymidine • extract DNA and stretch on glass slide • autoradiograph the fibers bi-directional uni-directional J Mol Biol. 1968 Mar 14;32(2):327-41. Replication is bi-directional • John Cairns, 1963; Huberman and Riggs, 1968 Experiment: Predictions: • pulse-label replicating cells with 3H-thymidine • then chase with cold thymidine • extract DNA and stretch on glass slide • autoradiograph the fibers bi-directional uni-directional J Mol Biol. 1968 Mar 14;32(2):327-41. enzymatic mechanism for the biosynthesis of deoxypolynucleotide in the 3' to 5' direction has been demonstrated, although 5' to 3' in vitro synthesis of DNA is accomplished semi-discontinuous Replication isby DNA polymerase.8 If discontinuous synthesis of DNA could by a reaction in occur in vivo (Figs. 1B-D), short stretches could be synthesized BIOCHEMISTRY: O • Reiji Okazaki, 1968 602 the 5' to 3' direction and subsequently connected to the growing polynucleotide chain by formation of phosphodiester linkages. 120w It is possible to distinguish between continuous s and discontinuous chain growth by elucidating B A f the structure of the most recently replicated C portion of the chromosome; that is, that portion , w A-1 lQOOO \taini 20,000by an extremely short radio/ 3' H o selectively labeled active pulse. E If the chromosome replicates disi f \ i )J 1 the mechanisms one of- 0X continuously byU60sec shown in _ D C Figures 1B, C, or D, a large portion of the _ p / z , \ radioactive label would be found in unconnected H Ah o 3 Ael A-' short chains which can be isolated, after deNT \an ° 10000 large J the naturation, from iOOO DNA chains derived t from the other portion of the chromosome. No R c molecular , Xadded FIG. 1.-Models for the possible such difference in the i 0w size between a structure and reaction in the the pulse-labeled and bulk DNA would be exw of replicating region of DNA. pected from a mechanism tk continuous synthesis 7w f .,,xY v < (Fig. 1A). t those reported previously,9 Our results to be described here, together with 0i 3 a 1 2system bacteriophage indicate that in a variety of bacterial systems and in one DISTANCE FROM TOP f most of the recently synthesized portion of the chromosome can be obtained Figure 6 Okazaki et al. Mechanism of DNA chain growth, I. Possible discontinuity after denaturation as small DNA molecules with a sedimentation coefficient of and unusual secondary structure of DNA chains. PNAS (1968) 59: 598. was recovered almost by which two about 10S. This supports the prediction of those mechanisms exclusively in DN Ol) enzymatic mechanism for the biosynthesis of deoxypolynucleotide in the 3' to 5' direction has been demonstrated, although 5' to 3' in vitro synthesis of DNA is accomplished semi-discontinuous Replication isby DNA polymerase.8 If discontinuous synthesis of DNA could by a reaction in occur in vivo (Figs. 1B-D), short stretches could be synthesized BIOCHEMISTRY: O • Reiji Okazaki, 1968 602 the 5' to 3' direction and subsequently connected to the growing polynucleotide chain by formation of phosphodiester linkages. 120w It is possible to distinguish between continuous 3 s • label cells with H-thymidine for short and discontinuous chain growth by elucidating B A f times to label newly synthesizedthe structure of the most recently replicated DNA C portion of the chromosome; that is, that portion , w A-1 lQOOO \taini 20,000by an extremely short radio/ • separate DNA by size 3' H o by selectively labeled ultracentrifugation in an ALKALINE active pulse. E If the chromosome replicates disi f \ sucrose gradient i )J 1 the mechanisms one of- 0X continuously byU60sec shown in _ D C Figures 1B, C, or D, a large portion of the _ p / z , \ • divide centrifuge tube into fractions radioactive label would be found in unconnected H Ah o 3 and count radioactivity in each short chains which can be isolated, after deAel A-' NT \an ° 10000 large J the naturation, from iOOO DNA chains derived t from the other portion of the chromosome. No R c molecular , Xadded FIG. 1.-Models for the possible such difference in the i 0w size between a structure and reaction in the the pulse-labeled and bulk DNA would be exw of replicating region of DNA. pected from a mechanism tk continuous synthesis 7w f .,,xY v < (Fig. 1A). t those reported previously,9 Our results to be described here, together with 0i 3 a 1 2system bacteriophage indicate that in a variety of bacterial systems and in one DISTANCE FROM TOP f most of the recently synthesized portion of the chromosome can be obtained Figure 6 Okazaki et al. Mechanism of DNA chain growth, I. Possible discontinuity after denaturation as small DNA molecules with a sedimentation coefficient of and unusual secondary structure of DNA chains. PNAS (1968) 59: 598. was recovered almost by which two about 10S. This supports the prediction of those mechanisms exclusively in DN Ol) enzymatic mechanism for the biosynthesis of deoxypolynucleotide in the 3' to 5' direction has been demonstrated, although 5' to 3' in vitro synthesis of DNA is accomplished semi-discontinuous Replication isby DNA polymerase.8 If discontinuous synthesis of DNA could by a reaction in occur in vivo (Figs. 1B-D), short stretches could be synthesized BIOCHEMISTRY: O • Reiji Okazaki, 1968 602 the 5' to 3' direction and subsequently connected to the growing polynucleotide chain by formation of phosphodiester linkages. 120w It is possible to distinguish between continuous 3 s • label cells with H-thymidine for short and discontinuous chain growth by elucidating B A f times to label newly synthesizedthe structure of the most recently replicated DNA C portion of the chromosome; that is, that portion , w A-1 lQOOO \taini 20,000by an extremely short radio/ • separate DNA by size 3' H o by selectively labeled ultracentrifugation in an ALKALINE active pulse. E If the chromosome replicates disi f \ sucrose gradient i )J 1 the mechanisms one of- 0X continuously byU60sec shown in _ D C Figures 1B, C, or D, a large portion of the _ p / z , \ • divide centrifuge tube into fractions radioactive label would be found in unconnected H Ah o 3 and count radioactivity in each short chains which can be isolated, after deAel A-' NT \an ° 10000 large J the naturation, from iOOO DNA chains derived t from the other portion of the chromosome. No R c molecular , Xadded FIG. 1.-Models for the possible such difference in the i 0w size between a structure and reaction in the the pulse-labeled and bulk DNA would be exw of replicating region of DNA. pected from a mechanism tk continuous synthesis 7w f .,,xY v < (Fig. 1A). t those reported previously,9 Our results to be described here, together with 0i 3 a 1 2system bacteriophage indicate that in a variety of bacterial systems and in one DISTANCE FROM TOP f most of the recently synthesized portion of the chromosome can be obtained Figure 6 Okazaki et al. Mechanism of DNA chain growth, I. Possible discontinuity after denaturation as small DNA molecules with a sedimentation coefficient of and unusual secondary structure of DNA chains. PNAS (1968) 59: 598. was recovered almost by which two about 10S. This supports the prediction of those mechanisms exclusively in DN Ol) Semi-conservative; Semi-discontinuous; bi-directional DNA Replication II: Multiple proteins collaborate to replicate DNA DNA polymerase cannot replicate the genome. 1. Can’t unwind double-stranded DNA 2. The single-stranded template is unstable 3. Can’t start DNA chains 4. Too slow and too distributive 5. No 3’ to 5’ polymerase 1. Can’t unwind double-stranded DNA Solution: DNA helicase 1. Can’t unwind double-stranded DNA Solution: DNA helicase Molecular Cell Volume 31, Issue 2, 25 July 2008, Pages 287-293 1. Can’t unwind double-stranded DNA Solution: DNA helicase T7 gene 4 protein E coli DnaB protein Molecular Cell Volume 31, Issue 2, 25 July 2008, Pages 287-293 SV40 T-Ag Sulfolobus solfataricus MCM The MCM (minichromosome maintenance) protein complex 3ʼ PNAS December 23, 2008 vol. 105 no. 51 20191-20196 J. Biol. Chem., Vol. 282, Issue 47, 34229-34234, November 23, 2007 2. The single-stranded template is unstable Solution: SSBs What happens to single-stranded DNA? The EMBO Journal (2001) 20, 612–618 The EMBO Journal (2002) 21, 1855–1863 ...
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This note was uploaded on 01/23/2012 for the course BCHM 311 taught by Professor Kelley during the Spring '09 term at University of Toronto- Toronto.

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