1 DNA: The Genetic Material Chapter 14

2 CH 14 Outline Chemical Nature of Nucleic Acids Three-Dimensional Structure of DNA – Watson and Crick Replication – Semi Conservative – Replication Process One-Gene/One-Polypeptide Hypothesis

3 Replication

4 DNA is the Genetic Material Therefore it must (1)Replicate faithfully. (2)Have the coding capacity to generate proteins and other products for all cellular functions. “A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.” -Francis Crick

5 The Dawn of Molecular Biology April 25, 1953 Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

6 Chemical Nature of Nucleic Acids DNA made up of nucleic acids – Each nucleotide is composed of a five carbon sugar, a phosphate group, and an organic base.  nucleotides distinguished by the bases  reaction between phosphate group of one nucleotide and hydroxyl group of another is dehydration synthesis  phosphodiester bond

7 Chemical Nature of Nucleic Acids Purines - large bases – adenine and guanine Pyrimidines - small bases – cytosine and thymine  Chargaff’s rule  A = T and G = C

8 Nucleotides

9 Three-Dimensional Structure of DNA X-ray diffraction suggested DNA had helical shape with a 2 nanometer diameter. – Watson and Crick deduced DNA is an inter- twined double helix.  complementary base-pairing  purines pairing with pyrimidines  constant 2 nanometer diameter  antiparallel configuration

10 DNA Double Helix

11 Bases paired Strands antiparallel

12 Models for DNA replication 1) Semiconservative model: Daughter DNA molecules contain one parental strand and one newly-replicated strand 2) Conservative model: Parent strands transfer information to an intermediate (?), then the intermediate gets copied. The parent helix is conserved, the daughter helix is completely new 3) Dispersive model: Parent helix is broken into fragments, dispersed, copied then assembled into two new helices. New and old DNA are completely dispersed

13 (a) Hypothesis 1: Semi-conservative replication (b) Hypothesis 2: Conservative replication Intermediate molecule (c) Hypothesis 3: Dispersive replication MODELS OF DNA REPLICATION

14 TEMPLATING/REPLICATION REPLICATION OF INFORMATION TEMPLATING

15 DNA replication Nucleotides are successively added using deoxynucleoside triphosphosphates (dNTP’s)

16 Key proposal of Watson and Crick: base pairs A : T and G : C are specific. Base pairing regulates replication.

17 DNA Replication Since DNA replication is semiconservative, therefore the helix must be unwound. John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an “origin” or “ori” for short.

18 Replication as a process Double-stranded DNA unwinds. The junction of the unwound molecules is a replication fork. A new strand is formed by pairing complementary bases with the old strand. Two molecules are made. Each has one new and one old DNA strand.

19 Replication can be Uni- or Bidirectional

20 Semi-Conservative Replication Each chain in the helix is a complimentary mirror image of the other. – double helix unzips and undergoes semi- conservative replication  each strand original duplex becomes one strand of another duplex  confirmed by Meselson-Stahl experiment

21 Replication Process Replication of DNA begins at one or more sites (replication origin). – DNA polymerase III and other enzymes add nucleotides to the growing complementary DNA strands.  require a primer  can only synthesize in one direction  endonucleases  exonucleases

22 DNA Replication

23 Replication Process DNA polymerase cannot link the first nucleotides in a newly synthesized strand. – RNA polymerase (primase) constructs an RNA primer. DNA polymerase adds nucleotides to 3’ end. – Leading strand replicates toward replication fork. – Lagging strand elongates from replication fork.  Okazaki fragments

24 DNA Synthesis

25 Replication Process DNA ligase attaches fragment to lagging strand. – Because synthesis of the leading strand is continuous and the lagging strand is discontinuous, the overall replication of DNA is referred to as semi-discontinuous. DNA gyrase removes torsional strain introduced by opening double helix.

26 Replication Process Opening DNA double helix – initiating replication – unwinding duplex – stabilizing single strands – relieving torque Building a primer Assembling complementary strands Removing the primer Joining Okazaki fragments

27 DNA Replication Fork

28 Replisome Replisome is a macromolecular protein machine (replication organelle). – fast, accurate replication of DNA during cell division

29 Stages of Replication Initiation – always occurs at the same site Elongation – majority of replication spent in elongation Termination – exact details unclear

30 Evidence points to bidirectional replication Label at both replication forks

31 Overview Features of DNA Replication DNA replication is semiconservative – Each strand of both replication forks is being copied. DNA replication is bidirectional – Bidirectional replication involves two replication forks, which move in opposite directions