Chapter 10: DNA structure and analysis

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Chapter 10 DNA structure and analysis
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Where were going

• The Central Dogma- DNA makes RNA makes Protein
• Evidence for DNA genetic material
• Structure of DNA- key words of polarity,
  complementary, antiparallel, knowing the bases.

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CENTRAL DOGMA OF MOLECULAR GENETICS

• DNA makes DNA (replication)
• DNA makes (carries the information to make)
• RNA (transcription)
• RNA makes (carries the information to make)
• PROTEIN (translation) which contributes to a
  particular
• TRAIT
• Fig. 10-1

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Convincing you that DNA is the genetic material

• Early- DNAboring, wrong, mislead a generation of
  scientists- just a scaffold for the more
  interesting proteins- a repeating A-T-C-G
  structure.
• Then, Chargaff- AT, GC, but the amounts of each
  type could vary quite a bit.
• I. DNA as the genetic material
  Avery, and Hershey/Chase,
• Avery showed that what Griffith found,
  transformation of rough Streptococcus
  ----gtsmooth, was caused by DNA. Fig 10-2,3
• The smooth phenotype was due to the presence of a
  capsule- made Strep pneumoniae resistant to
  phagocytosis.

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DNA as the genetic material Avery, and
Hershey/Chase

• I. Avery showed that what Griffith found,
  transformation of rough Streptococcus
  ----gtsmooth, was caused by DNA. Fig 10-2,3
• The smooth phenotype was due to the presence of a
  capsule- made Strep pneumoniae resistant to
  phagocytosis.

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Hershey-Chase

• Bacteriophage infect kill cells
• Known to inject material into cells- not the
  whole virus- so the injected material had the
  information to make a virus.
• Protein or DNA???

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II. Structure of DNA Linear, double-stranded
chains of deoxynucleotides

• deoxynucleotides
• nitrogenous bases Adenine, thymine, cytosine,
  guanine AT, GC
• The chains have polarity because of the linkage-
  a 5' and 3' end to each molecule.
• LO Be able to recognize the bases found in DNA
  and RNA, and the partners to which they pair.
• Distinguish between a nucleoside
  nucleotide

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1 ring, big name
2 rings, small name
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• Learning Objective What is the structure of
  DNA? Specifically
• Understand the polarity of DNA and its
  antiparallel nature
• given a sequence, be able to give its
  complementary sequence.
• Be able to identify the major and minor
  grooves of DNA.
• Know what is meant by DNA being a right-handed
  helix, and by
• supercoiling.
• Be able to identify the coding and
  anticoding strands of DNA
• when it is used as a template for transcription.

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Nucleotides are linked together- POLARITY!
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Six mistakes! Find 3!
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2 nm
3.4 nm, 0.34 nm/base
10 bp/turn
Rt handed dbl helix, complementary, antiparallel
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How I test this

• 5' ATGACCTTAGG3- give the complementary strand,
  RNA or DNA

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Features that make DNA suitable as genetic
material

• a. Strands are complementary thus, each
  strand is the template, holds the information,
  for the other strand the pattern for replication
  is built-in.
• b. The bases allow for a three-base code
  there are 64 possible combinations of three
  bases, more than enough to code for all the amino
  acids.
• c. The structure allows for both faithful
  duplication, and for mutations that will then be
  perpetuated.

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Structure of RNA

• Ribose and uracil, not deoxyribose and thymine.
• usually single stranded
• can fold, to produce secondary structure
• Three main types, but there are others rRNA,
  mRNA, tRNA

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III. Neat things you can do with DNA, and what
it means

• A. Denaturation and renaturation DNA can be
  denatured- rendered SS- by heat or NaOH
  treatment. HOWEVER upon heating to 68C, it will
  renature- find its complementary bases and reform
  DS DNA.
• B. hybridization Because DNA can renature, you
  can prepare probes labeled DNA that will
  hybridize to unlabeled target DNA, either in
  solution, or when the target is immobilized to
  paper. A probe will find its complementary DNA
  rapidly, even in the midst of a vast excess of
  non-target DNA. One application FISH (10-15).

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C. Determining its size

• Gel electrophoresis small DNA fragments migrate
  faster than large rate is an inverse log
  function. Fig 10-19
• Pulsed field gel electrophoresis variation of
  electrophoresis that can separate large fragments
  of DNA.
• Electron microscopy Opening figure of Ch. 11

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What to know

• See the learning objectives! But specifically
• Evidence for DNA being genetic material Avery
  Hershey/Chase
• Structure (including components), w/ key words
  (rt handed helix, complementary, polarity,
  antiparallel, etc.)
• Denaturation, hybridization, size determination.

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Chapter 11- DNA replication recombination

• Check out the how do we know section
• I. Replication is
• SEMICONSERVATIVE
• BIDIRECTIONAL
• SEMIDISCONTINUOUS

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• A. Semiconservative Fig. 11.3, 11.4
• Proven by Messelsohn-Stahl experiment Heavy
  (15N) DNA-----gt HL, then LL,
• with a fixed amount of HL remaining. Separation
  is by CsCl density gradient centrifugaton. (Also
  Taylor, Woods, Hughes)

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• B. BIDIRECTIONAL Figs 11-6 Low 3H thymidine
  pulse is followed by high 3H thymidine pulse
  results after autoradiography is a set of
  symmetrical dark bands.
• Bacterial spores-? germinate, resulting in
  synchronous initiation of DNA replication? pulse
  with low 3H thymidine pulse? high 3H thymidine
  pulse? autoradiography. The results show
  symmetrical lines of thymidine incorporation into
  the DNA (See Q. 32 in your text)

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• C. Semidiscontinuous Fig. 11.11 One strand is
  made continuously, one strand discontinuously
• Well cover evidence a bit later.

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• II. Process of replication AS WITH ALL
  MACROMOLECULAR SYNTHESIS INITIATION,
  ELONGATION, TERMINATION.
• Cool video of the process- an animation
• http//www.wehi.edu.au/education/wehi-tv/dna/repli
  cation.html

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• The process of DNA replication is driven by 1)
  antiparallel nature of DNA 2) the nature of DNA
  polymerases a) ONLY elongate from a 3'-OH, i.e.,
  only replicate in a 5'-3'direction DO NOT
  initiate, ONLY elongate.
• Bacterial DNA polymerases are VERY fast 1000
  bp/sec!
• 5'---------------T3'OH pppdG3'OH
• 3'---------------ACGGATCGAGAG-----------------5'
• 5'---------------TG3'OH pppdC3'OH
• 3'---------------ACGGATCGAGAG-----------------5'
• 5'---------------TGC3'OH pppdC3'OH
• 3'---------------ACGGATCGAGAG-----------------5'
• etc.

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• A. INITIATION BEGINS at a particular location,
  the origin- signaled by the cell. Prokaryotes
  have a single origin, eukaryotes have many.
  Replication is initiated by an increase in cell
  mass, triggered by signals received from the
  cell. Initiation proteins open up the helix at
  the origin. Key protein Dna A. Fig. 11-9. A
  region replicated by a single origin is a
  replicon. Bacteria usually have one, euk. have
  many hundreds of replicons.

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Origin region
A helicase and loader of primase
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• B. ELONGATION once synthesis has begun, it
  usually proceeds bidirectionally. Because
  synthesis is always 5'-3', synthesis tends to be
  CONTINUOUS on one strand of a replication fork,
  and DISCONTINUOUS on the other strand of the
  fork. Primase, with the help of the mobile
  promoter helicase, the DNA BC complex, moves
  down the lagging strand, laying down primer for
  DNA pol to use. Lagging strand synthesis
  produces short fragments of 1-2K bases, called
  Okazaki fragments. (Fig 11-11)

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• C. HOW WE KNOW THIS Labeling experiments with
  replicating viruses if you label replicating DNA
  with a short pulse of radioactivity, you will
  label short (Okazaki) fragments, and longer
  continuous strand fragments, that can be
  separated by an alkaline sucrose density gradient
  (crude blackboard drawing here)

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• D. Other players Helicase again, the DnaBC
  complex helps primase get started, and also
  separates the strands to allow replication DNA
  gyrase Introduces negative supercoils, acting to
  allow the DNA to swivel, preventing overwinding
  of the helix. Theres also a single-stranded
  binding protein (SSBP) that, well, binds single
  stranded DNA- keeping it SS as needed.

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• E. Completing the job the problem of ends.
• Fig. 11-16, 17 circles do not present a problem
  for termination two circles are made. Linear
  DNA does present a problem, b/c of the gap left
  by the lack of primer at the 5' end of the new
  DNA. In Eukaryotes, the problem is solved by
  TELOMERASE.

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• Other forms of replication rolling circle
  Some viruses replicate from a nick in the DNA
  the new DNA unwinds one parental strand, that
  then replicates by lagging strand synthesis.
  This produces a CONCATEMER of DNA that is then
  processed.

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• Proofreading fidelity of replication is enhanced
  by proofreading. If the wrong base is put in,
  the mismatch is removed before replication
  continues. This enhances the accuracy of
  replication.
• pppdG3'OH
• 5'--------------AC3'OH
• 3'--------------TACGGATCGAGAG-----------------5'
• Oops! MISMATCH!!!!
• 3-5 exonuclease activity removes mismatch
• 5'-------------AC3'OH
• 3'-------------TACGGATCGAGAG-----------------5'
• pppdT3'OH
• 5'------------- A3'OH pdC3OH
• 3'--------------TAGGATCGAGAG-----------------5'
• Replication continues

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Recombination What, Why and how

• What breaking and rejoining two pieces of DNA
• A, a similar double-stranded DNA!!
• AAAAAAAAAAAAAAAAAAAAAAAA -gt
• Aaaaaaaaaaaaaaaaaaaaaaaaaaaaa usually two
  similar strands
• AAAAAAAAAAaaaaaaaaaaaaaaaaaa
• aaaaaaaaaaaaAAAAAAAAAAAAAA
• Or sometimes an insertion
• AAAAAAAAAAAAAAAAAAAAAAAA BBBBBB -gt
• AAAAAAAAAA BBBBBB AAAAAAAAAAAAAA

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What to know

• The key players
• Put it together for a replication fork.
• Evidence for semiconservative, bidirectional,
  semidiscontinuous.

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• Why 1) promotes genetic exchange (so- why should
  an organism want this??)
• 2) Repair Rec- mutants in bacteria are UV
  sensitive, die easily, dont mutagenize well- the
  major reason for a cell.

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Three types

• 1. Site specific 2 regions of short homology
• 20 bp of homology
• -----
• -------------------------------------------
  -------
• Some viruses integrate into the host chromosome
  this way

20 bp of homology
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• 2. Illegitimate Transposons can insert
  anywhere

Randomly inserting Tns
E. Coli chromosome
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3. Homologous recombination the main type.

• HOW of homologous fig 11-18

Its not really quite this neat- probably w
different nicks, but same net effect.
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Gene Conversion and Mismatch Repair

• Def One allele is converted to another. Thought
  to occur during heteroduplex formation in
  recombination, followed by mismatch repair. Fig.
  11-19.
• AA -gt at
  heteroduplex
• aa
• aA
• aA
• Repair converts one allele to another
• AA
• aA

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Things to know

• You should be able to put all of the information
  about the DNA replication proteins into a model
  of a replication fork for DNA- tell the story of
  DNA replication.
• 2. What is meant by bi-and uni-directional
  replication, and how whats the evidenceit shown?
  What is a replicon?
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• 3. What is meant by semi-discontinuous
  replication, and how is it shown?
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Things to know (contd)

• 4. What is meant by rolling circle replication?
• Why is duplicating the ends of a linear molecule
  a problem, what is one solution (in particular,
  telomerase as a solution)
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• 5. Why do genes recombine? What are some of the
  possible mechanisms for recombination? How does
  recombination explain gene conversion? (STORY!)
•