The Molecular Basis of Inheritance

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Chapter 16

• The Molecular Basis of Inheritance

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But first Bessbugs! (Bess beetles)
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Overview Lifes Operating Instructions

• In 1953, Watson and Crick introduced an elegant
  double-helical model for the structure of
  deoxyribonucleic acid, or DNA
• Hereditary information is encoded in DNA and
  reproduced in all cells of the body
• This DNA program
  directs the development
  
  of biochemical, anatomical,
  
  physiological, and
  (to some
  extent)
  behavioral traits

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Nucleic acid
1869 Friedrich
Miescher isolated DNA from pus!
DNA and RNA are nucleic acids.
Each nucleotide consists of a 1. pentose
sugar 2. nitrogenous base 3. phosphate group
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Sugarphosphatebackbone
Nitrogenous bases
5? end
Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate
Guanine (G)
Sugar(deoxyribose)
DNA nucleotide
Nitrogenous base
3? end
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What is the genetic material?

• When genes were shown to be located on
  chromosomes, the 2 components of chromosomes- DNA
  and protein- became
  candidates for the genetic material
• Protein seemed more complex than DNA
• The key factor in determining the genetic
  material was choosing appropriate experimental
  organisms
• The role of DNA in heredity was first discovered
  by studying bacteria and the viruses that infect
  them

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Evidence That DNA Can Transform Bacteria

• Discovery of genetic role of DNA began w/
  research by Frederick Griffith in 1928,
• who worked with two strains of a bacterium,
  Streptococcus pneumoniae
• pathogenic S (smooth) strain
• harmless R (rough) strain

• When he mixed heat-killed remains of the
  pathogenic strain with living cells of the
  harmless strain, some living cells became
  pathogenic
• He called this phenomenon transformation,
• now defined as a change in genotype and
  phenotype due to assimilation of foreign DNA

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Evidence That Viral DNA Can Program Cells

• In 1944, Oswald Avery, Maclyn McCarty, and Colin
  MacLeod announced that the transforming substance
  was DNA
• Avery-MacLeod-McCarty experiment
• More evidence for DNA as the genetic material
  came from studies of a virus that infects
  bacteria
• Such viruses, called bacteriophages (or phages),
• are widely used in molecular genetics research

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In 1952, Hershey and Chase performed experiments
showing that only one of the two components (DNA/
protein) of a phage T2
enters an E. coli cell during infection
In 1952, Hershey and Chase performed experiments
showing that only one of the two components (DNA/
protein) of a phage T2
enters an E. coli cell during infection
In 1952, Hershey and Chase performed experiments
showing that only one of the two components (DNA/
protein) of a phage T2
enters an E. coli cell during infection
Empty protein shell
Empty protein shell
Radioactivity (phage protein)
in liquid
Radioactive protein
Radioactive protein
Radioactive protein
Phage
Phage
Phage
Bacterial cell
Bacterial cell
Bacterial cell
DNA
DNA
DNA
Batch 1 radioactive sulfur (35S)
Batch 1 radioactive sulfur (35S)
Batch 1 radioactive sulfur (35S)
Phage DNA
Phage DNA
radioactive sulfur labels protein
radioactive sulfur labels protein
radioactive sulfur labels protein
Centrifuge
shake loose phages that remained outside the
bacteria
shake loose phages that remained outside the
bacteria
Pellet (bacterial cells and contents)
Radioactive DNA
Radioactive DNA
Radioactive DNA
Batch 2 radioactive phosphorus (32P)
Batch 2 radioactive phosphorus (32P)
Batch 2 radioactive phosphorus (32P)
radioactive phosphorus labels DNA
radioactive phosphorus labels DNA
radioactive phosphorus labels DNA
Centrifuge
Radioactivity (phage DNA) in pellet
Pellet
They concluded that the injected DNA of the
phage provides the genetic information
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Building a Structural Model of DNAClue 1
Erwin Chargaff
A
T
G
C
In 1950, reported that DNA composition varies
from one species to the next
Chargaffs rules state that in any species there
is an equal number of A and T bases, and an equal
number of G and C bases

• This evidence of diversity made DNA a more
  credible candidate for the genetic material

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Building a Structural Model of DNAClue 2

• Wilkins and Franklin were using a technique
  called
• X-ray crystallography to study molecular
  structure

• Franklin produced a picture of the DNA molecule
  using this technique

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Rosalind Franklin (1920-1958)
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• The X-ray crystallographic images of DNA enabled
  Watson to deduce that
• 1. DNA was helical
• 2. the width of the helix and the spacing of
  the nitrogenous bases
• suggested that the DNA molecule was made up of
  two strands,
  forming a double helix

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• At first, Watson and Crick thought the bases
  paired like
  with like (A with A, and so on), but such
  pairings did not result in a uniform width
• Instead, pairing a purine with a pyrimidine
  resulted in a uniform width consistent with the
  X-ray

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They determined adenine (A) paired only with
thymine (T) guanine (G) paired only with
cytosine (C)
2 hydrogen bonds
Sugar
Sugar
Adenine (A)
Thymine (T)
Sugar
3 hydrogen bonds
Sugar
Guanine (G)
Cytosine (C)
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DNA Replication

• Watson and Crick noted that the specific base
  pairing suggested a
  possible copying mechanism for genetic material
• Since the two strands of DNA are complementary,
  each
  strand acts as a template for building a new
  strand in replication
• In DNA replication, the parent molecule unwinds,
  and 2
  new daughter strands are built based on
  base-pairing rules

T
T
A
T
A
T
A
T
A
T
A
T
A
T
A
A
T
A
T
A
T
A
A
T
A
T
G
G
C
G
C
G
C
G
C
G
C
G
C
G
C
C
G
C
G
C
G
C
C
G
C
G
A
A
T
A
T
A
T
A
T
A
T
A
T
A
T
A
T
A
T
A
T
A
T
A
T
T
T
T
T
T
T
T
T
T
T
T
T
A
A
A
A
A
A
T
A
A
A
T
A
A
A
A
G
C
G
C
G
C
G
C
G
G
C
C
C
G
C
G
G
C
C
C
G
C
G
C
G
G
The first step in replication is separation of
the two DNA strands.
The first step in replication is separation of
the two DNA strands.
The first step in replication is separation of
the two DNA strands.
The nucleotides are connected to form
the sugar-phosphate back- bones of the new
strands. Each daughter DNA molecule consists of
one parental strand of one new strand.
Each parental strand now serves as a template
that determines the order of nucleotides along a
new, complementary strand.
Each parental strand now serves as a template
that determines the order of nucleotides along a
new, complementary strand.
The parent molecule has two complementary strands
of DNA. Each base is paired by hydrogen bonding
with its specific partner, A with T and G with C.
The parent molecule has two complementary strands
of DNA. Each base is paired by hydrogen bonding
with its specific partner, A with T and G with C.
The parent molecule has two complementary strands
of DNA. Each base is paired by hydrogen bonding
with its specific partner, A with T and G with C.
The parent molecule has two complementary strands
of DNA. Each base is paired by hydrogen bonding
with its specific partner, A with T and G with C.
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April 23, 2012
19

• Updated syllabus
• Homework

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Ch 16 so far

• The history
• Griffith (1928)
• Avery-MacLeod-McCarty (1944)
• Hershey and Chase (1952)
• Francis and Crick (and Franklin) (1953)
• Meselson and Stahl (1958)
• DNA Replication
• Chromosome molecular structure

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First replication
Second replication

• Watson and Cricks
• semiconservative
• model
• of replication predicts that when a
  double helix replicates, each daughter molecule
  will have one old strand
  (derived or conserved
  from the parent molecule)
  and one newly
  made strand
• Competing models were the
  conservative model the dispersive
  model

Parent cell
Conservative model. The two parental strands
reassociate after acting as templates for new
strands, thus restoring the parental double helix.
Semiconservative model. The two strands of the
parental molecule separate, and each functions
as a template for synthesis of a new,
comple-mentary strand.
Dispersive model. Each strand of both daughter
molecules contains a mixture of old and newly
synthesized DNA.
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Bacteria cultured in medium containing 15N
Bacteria transferred to medium containing 14N
Meselson Stahl- To test the 3 models they
labeled the nucleotides of the old strands with a
heavy isotope of nitrogen,
while any new nucleotides were labeled with a
lighter isotope The first
replication produced a band of hybrid DNA,
eliminating the conservative
model A second replication produced both light
and hybrid DNA, eliminating the dispersive model
and supporting the semiconservative model
light (14N)
heavy (15N)
light (14N)
Less dense
DNA sample centrifuged after 20 min (after
first replication)
DNA sample centrifuged after 40 min (after
second replication)
hybrid
hybrid
More dense
First replication
Second replication
Conservative model
light (14N)
light (14N)
heavy (15N)
heavy (15N)
Semiconservative model
light (14N)
hybrid
hybrid
Dispersive model
mostly light (14N)
hybrid
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DNA replication overview
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Origin of replication
Parental (template) strand

• DNA Replication begins at special sites called
• origins of replication,
• A eukaryotic chromosome may have 100-1,000s
• The two DNA strands are separated,
  opening up a
• replication bubble
• consisting of 2
• replication forks,
• Y-shaped regions where new DNA strands are
  elongating at each end

Daughter (new) strand
Replication fork
Double- stranded DNA molecule
Replication bubble
0.5 µm
Two daughter DNA molecules
(a) Origins of replication in E. coli
replication fork
Origin of replication
Double-stranded DNA molecule
Parental (template) strand
Daughter (new) strand
0.25 µm
Replication fork
Bubble
Two daughter DNA molecules
(b) Origins of replication in eukaryotes
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• Helicase-
• untwists the double helix and separates the
  template DNA strands at the replication fork
• Single-strand binding protein-
• binds to and stabilizes single-stranded DNA
  until it can be used as a template
• Topoisomerase-
• corrects overwinding ahead of replication
  forks by breaking, swiveling, rejoining DNA
  strands

Single-strand binding proteins
RNA primer (5-10 nucleotides)
3?
Topoisomerase
5?
3?
Primase- synthesizes the initial nucleotide
strand, a short RNA primer
5?
5?
3?
Helicase
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Elongating a New DNA Strand

• The rate of elongation is 500 nucleotides/ sec
  in bacteria 50 nucleotides/ sec in human cells

New strand
Template strand
3 end
5 end
5 end
3 end
enzymes that catalyze the elongation of new DNA
at a replication fork
Sugar
Base
Phosphate
DNA polymerase
3 end
Each nucleotide that is added to a growing DNA
strand is a nucleoside triphosphate
3 end
Pyrophosphate
Nucleoside triphosphate
5 end
5 end
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Antiparallel Elongation

• The antiparallel structure of the double helix
  (two strands
  oriented in opposite directions)
  affects replication
• DNA polymerases add nucleotides only to the
  free 3??end of a growing
  strand
  therefore, a new DNA strand
  can
  elongate only in the 5??to 3??direction

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Overview

• Along one template strand of DNA, called the
  leading strand,
• DNA polymerase can synthesize a
  complementary strand continuously,
  moving toward the replication fork

Origin of replication
Leading strand
Lagging strand
5?
3?
Primer
5?
3?
Leading strand
Lagging strand
Overall directions of replication
Origin of replication
3?
5?
RNA primer
5?
Sliding clamp
3?
5?
DNA poll III
Parental DNA
3?
5?
5?
3?
5?
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To elongate the other new strand,
called the lagging strand, DNA polymerase must
work in the direction away from the replication
fork The lagging strand is synthesized as a
series of segments called Okazaki
fragments, which are joined together by DNA
ligase
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Synthesis of the lagging strand
1
1
1
1
1
Primase joins RNA nucleotides into a primer.
Primase joins RNA nucleotides into a primer.
Primase joins RNA nucleotides into a primer.
Primase joins RNA nucleotides into a primer.
1
Primase joins RNA nucleotides into a primer.
Primase joins RNA nucleotides into a primer.
3?
3?
3?
3?
3?
3?
5?
5?
5?
5?
5?
5?
3?
3?
3?
3?
3?
5?
5?
5?
5?
5?
3?
5?
Templatestrand
Templatestrand
Templatestrand
Templatestrand
Templatestrand
Templatestrand
2
2
2
2
DNA pol III adds DNA nucleotides to the primer,
forming an Okazaki fragment.
DNA pol III adds DNA nucleotides to the primer,
forming an Okazaki fragment.
DNA pol III adds DNA nucleotides to the primer,
forming an Okazaki fragment.
2
DNA pol III adds DNA nucleotides to the primer,
forming an Okazaki fragment.
DNA pol III adds DNA nucleotides to the primer,
forming an Okazaki fragment.
3?
3?
3?
3?
3?
5?
5?
5?
5?
5?
3?
RNA primer
3?
RNA primer
RNA primer
3?
3?
RNA primer
3?
RNA primer
5?
5?
5?
5?
5?
3
3
3
3
After reaching the next RNA primer (not shown),
DNA pol III falls off.
After reaching the next RNA primer (not shown),
DNA pol III falls off.
After reaching the next RNA primer (not shown),
DNA pol III falls off.
After reaching the next RNA primer (not shown),
DNA pol III falls off.
Okazakifragment
Okazakifragment
Okazakifragment
Okazakifragment
3?
3?
3?
3?
3?
3?
3?
3?
5?
5?
5?
5?
5?
5?
5?
5?
4
4
After the second fragment is primed, DNA pol III
adds DNAnucleotides until it reaches the first
primer and falls off.
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After the second fragment is primed, DNA pol III
adds DNAnucleotides until it reaches the first
primer and falls off.
After the second fragment is primed, DNA pol III
adds DNAnucleotides until it reaches the first
primer and falls off.
5?
5?
5?
3?
3?
3?
3?
3?
3?
5?
5?
5?
5
5
DNA pol 1 replaces the RNA with DNA, adding to
the 3? end of fragment 2.
DNA pol 1 replaces the RNA with DNA, adding to
the 3? end of fragment 2.
5?
5?
3?
3?
3?
3?
5?
5?
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The lagging strand in this region is
nowcomplete.
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DNA ligase forms a bond between the newest
DNAand the adjacent DNA of fragment 1.
5?
3?
3?
5?
Overall direction of replication
Overall direction of replication
Overall direction of replication
Overall direction of replication
Overall direction of replication
Overall direction of replication
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REVIEW
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April 25, 2012
Self Description
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Announcements

• Homework due tomorrow (Apr 26)
• Old exams
• Exam III to hand back at the end of class

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Overview
Origin of replication
Lagging strand
Leading strand
Leading strand
Lagging strand
Single-strand binding protein
Overall directions of replication
Helicase
Leading strand
DNA pol III
5?
3?
3?
Primer
Primase
5?
Parental DNA
3?
Lagging strand
DNA pol III
5?
DNA pol I
DNA ligase
4
3?
5?
3
1
2
3?
5?
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The DNA Replication Machine

• The proteins that participate in DNA replication
  form a large complex, a DNA replication
  machine
• is probably stationary during the replication
  process
• Recent studies support
  
  a model in which
  DNA
  polymerase molecules reel in
  
  
  parental DNA and
  
  extrude newly made
  
  daughter DNA
  molecules

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Proofreading Repairing DNA
nucleotide excision repair
A thymine dimer distorts the DNA molecule.
often caused by UV

• DNA polymerases proofread newly made DNA,
  replacing any incorrect nucleotides
• DNA can be damaged by
• chemicals
• X-rays
• UV light
• toxic chemicals- cigarettes
• mismatch repair of DNA-
• repair enzymes correct errors in base pairing
• nucleotide excision repair-
• enzymes cut out and replace damaged stretches of
  DNA

A nuclease enzyme cuts the damaged DNA
strand at two points and the damaged section
is removed.
Nuclease
Repair synthesis by a DNA polymerase fills
in the missing nucleotides.
DNA polymerase
DNA ligase
DNA ligase seals the free end of the new
DNA to the old DNA, making the strand complete.
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Replicating the Ends of
DNA Molecules
5
Leading strand
End of parental DNA strands
Lagging strand
3
Last fragment
Previous fragment
RNA primer

• Limitations of DNA polymerase create problems for
  the linear DNA of eukaryotic chromosomes
• The usual replication machinery provides no way
  to complete the 5? ends, so repeated
  rounds of replication produce
  shorter DNA molecules with uneven ends

Lagging strand
5
3
Primer removed but cannot be replaced with DNA
because no 3 end available for DNA polymerase
Removal of primers and replacement with DNA where
a 3 end is available
5
3
Second round of replication
5
3
New leading strand
5
New leading strand
3
Further rounds of replication
Shorter and shorter daughter molecules
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• Eukaryotic chromosomal DNA molecules have at
  their ends nucleotide sequences called
• telomeres
• Telomeres do not prevent the shortening of DNA
  molecules, but they do postpone the erosion of
  genes near the ends of DNA molecules

TTAGGG 100 - 1,000 x
telomeres
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• If chromosomes of germ cells became shorter in
  every cell cycle, essential genes would
  eventually be missing from gametes they produce
• An enzyme called telomerase
  
• catalyzes the lengthening of telomeres in germ
  cells
• There is evidence of telomerase activity in
  cancer cells,
  which may allow cancer cells to persist
• It has been proposed that the shortening of
  telomeres is connected to
  aging
• The shortening of telomeres might protect cells
  from cancerous
  growth by limiting the number of cell divisions

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Chromosome structure

• The bacterial chromosome is a double-stranded,
  circular DNA molecule associated with a small
  amount of protein
• Eukaryotic chromosomes have linear DNA molecules
  associated with a large amount of protein
• Chromatin is a complex of DNA and protein, and is
  found in the nucleus of eukaryotic cells
• Histones are proteins that are responsible for
  the first level of DNA packing in chromatin

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Nucleosome (10 nm in diameter)
DNA double helix
(2 nm in diameter)
H1
Histone tail
Histones
DNA, the double helix
Histones
Nucleosomes, or beads on
a string (10-nm fiber)

• DNA winds around histones to form nucleosome
  beads
• Nucleosomes are strung together like beads on a
  string by linker DNA

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Chromatid (700 nm)
30-nm fiber
Loops
Scaffold
300-nm fiber
Interactions between nucleosomes cause the thin
fiber to coil or fold into this thicker fiber
Replicated chromosome (1,400 nm)
The 30-nm fiber forms looped domains that attach
to proteins
30-nm fiber
Metaphase chromosome
Looped domains (300-nm fiber)
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• Most chromatin is loosely packed in the nucleus
  during interphase and condenses prior to mitosis
• euchromatin-
• loosely packed chromatin
• heterochromatin-
• highly condensed chromatin
• during interphase a few regions of chromatin
  (centromeres and telomeres)
• Dense packing of the heterochromatin makes it
  difficult for the cell to express genetic
  information coded in these regions

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Painted Chromosomes

• At interphase, some chromatin is organized into a
  10-nm fiber, but much is compacted into a
  30-nm fiber, through folding and looping
• Interphase chromosomes are not highly condensed,
  but still occupy specific restricted regions in
  nucleus