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  • Advanced Cell Biology. Lecture 12

    Advanced Cell Biology. Lecture 12

    Alexey Shipunov

    Minot State University

    February 10, 2012

  • Advanced Cell Biology. Lecture 12

    Outline

    Questions and answers

    ProteinsHow proteins are studied

    DNADNA replication

  • Advanced Cell Biology. Lecture 12

    Outline

    Questions and answers

    ProteinsHow proteins are studied

    DNADNA replication

  • Advanced Cell Biology. Lecture 12

    Outline

    Questions and answers

    ProteinsHow proteins are studied

    DNADNA replication

  • Advanced Cell Biology. Lecture 12

    Questions and answers

    Previous final question: the answer

    Which way of sedimentation employs differences betweenmolecular sizes?

    I Velocity sedimentation: larger proteins sediment faster

  • Advanced Cell Biology. Lecture 12

    Questions and answers

    Previous final question: the answer

    Which way of sedimentation employs differences betweenmolecular sizes?

    I Velocity sedimentation: larger proteins sediment faster

  • Advanced Cell Biology. Lecture 12

    Questions and answers

    Teams

    I Studies on the chemical nature of the substance inducingtransformation of Pneumococcal types. — Avery, O.T.,MacLeod, C.M. & McCarty, M.J. — Exp. Med. 79, 137-159(1944)

    I Molecular Structure of Deoxypentose Nucleic Acids —Wilkins M.H.F., A.R. Stokes A.R. & Wilson, H.R. —Nature171, 738-740 (1953)

    I (1) A Structure for Deoxyribose Nucleic Acid — WatsonJ.D. and Crick F.H.C. — Nature 171, 737-738 (1953) AND(2) Genetical Implications of the structure ofDeoxyribonucleic Acid — Watson J.D. and Crick F.H.C. —Nature 171, 964-967 (1953)

  • Advanced Cell Biology. Lecture 12

    Proteins

    How proteins are studied

    ProteinsHow proteins are studied

  • Advanced Cell Biology. Lecture 12

    Proteins

    How proteins are studied

    X-rays crystallography

    I Based on the ability of proteins to form crystalsI These crystals will scatter X-rays and we will see difraction

    pattern, different for different proteinsI Useful for prediction of protein 3D structure

  • Advanced Cell Biology. Lecture 12

    Proteins

    How proteins are studied

    X-rays crystallography

  • Advanced Cell Biology. Lecture 12

    Proteins

    How proteins are studied

    Nuclear magnetic resonance (NMR)

    I Unfortunately, not all proteins form crystals and it is notalways possible to obtain enough protein for crystallization

    I Method is based on the response of nuclei to radio wavesI Again, it is used mostly for understanding conformation of

    protein

  • Advanced Cell Biology. Lecture 12

    Proteins

    How proteins are studied

    Nuclear magnetic resonance

  • Advanced Cell Biology. Lecture 12

    Proteins

    How proteins are studied

    Protein databases

    I UniProtI NCBI proteinI RCSB

  • Advanced Cell Biology. Lecture 12

    Proteins

    How proteins are studied

    NCBI Protein database

  • Advanced Cell Biology. Lecture 12

    Proteins

    How proteins are studied

    RCSB database

  • Advanced Cell Biology. Lecture 12

    Proteins

    How proteins are studied

    UniProt database

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    DNADNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    Template

    I Both DNA strands may act as a template for the synthesisof other strand

    I Template hypothesis was first expressed by Nikolaj Koltsovin 1927

    I Replication is semiconservative

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    Meselson-Stahl experiment (1958)

    I Theoretically, three variants of replications were possible:semiconservative, dispersive and conservative

    I Experiment was based on two bacterial cultures whichgrew on different media: with normal nitrogen 14N, andwith heavy nitrogen, 15N

    I After growing for 20’ on heavy medium, bacteria produceDNA molecules with intermediate weight only

    I That ruled out conservative hypothesis.

    How to rule outdispersive hypothesis?—Explanation has been given onlecture.

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    Meselson-Stahl experiment (1958)

    I Theoretically, three variants of replications were possible:semiconservative, dispersive and conservative

    I Experiment was based on two bacterial cultures whichgrew on different media: with normal nitrogen 14N, andwith heavy nitrogen, 15N

    I After growing for 20’ on heavy medium, bacteria produceDNA molecules with intermediate weight only

    I That ruled out conservative hypothesis. How to rule outdispersive hypothesis?—Explanation has been given onlecture.

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    Replication origin and forks

    I DNA double helix should open for replication: this is areplication origin place

    I When replication starts, these openings will grow and formreplication forks which are visible under microscope

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    Lagging and leading strands

    I DNA is synthesized only in 5’-to-3’ direction. Structureof DNA and complexity of replication do not allow the otherdirection.

    I Therefore, fork is asymmetrical: one strand is replicatingsmoothly whereas other strand (lagging) replicated by“leaps”, still 5’-to-3’

    I Every “leap” produce one Okazaki fragment which are laterjoined

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    DNA proofreading

    I Every time DNA polymerase adds new nucleotide, itchecks if previous was correctly placed (if not, it removesthe wrong nucleotide)

    I Two different binding sites in DNA polymerase work forsynthesis and proofreading

    I Proofreading goes in opposite, 3’-to-5’ directionI As a result, DNA polymerase enzyme error rate became

    less than ≈0.00000001

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    DNA polymerase movie

    mov/dna_polymerase.wmv

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    RNA primers

    I DNA polymerase cannot start nucleotide chain itselfI Instead, primase enzyme synthesize small RNA primer

    (≈10 nucleotides) which used as a starting pointI Primase error rate is ≈0.0001 (!)I In lagging strand, RNA sites are interleaving with DNA

    fragmentsI Then RNA sites are erasing, and DNA ligase joins

    fragments together

    I Why primer is RNA?

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    RNA primers

    I DNA polymerase cannot start nucleotide chain itselfI Instead, primase enzyme synthesize small RNA primer

    (≈10 nucleotides) which used as a starting pointI Primase error rate is ≈0.0001 (!)I In lagging strand, RNA sites are interleaving with DNA

    fragmentsI Then RNA sites are erasing, and DNA ligase joins

    fragments togetherI Why primer is RNA?

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    Final question (3 points)

    Why cells use RNA as DNA replication primers?

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    Final question (3 points)

    Why cells use RNA as DNA replication primers?

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    Summary

    I DNA replication is a semiconservative processI DNA replication could go only in one directionI Proofreading and RNA priming are helping in replication

  • Advanced Cell Biology. Lecture 12

    DNA

    DNA replication

    For Further Reading

    A. Shipunov.Advanced Cell Biology [Electronic resource].2011—onwards.Mode of access: http://ashipunov.info/shipunov/school/biol_250

    B. Alberts et al.Essential Cell Biology. 3rd edition.Garland Science, 2009.Chapter 6, pages 198–208.

    http://ashipunov.info/shipunov/school/biol_250http://ashipunov.info/shipunov/school/biol_250

    Questions and answersProteinsHow proteins are studied

    DNADNA replication