Molecular Basis of Inheritance

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Chapter 16

• Molecular Basis of Inheritance

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Historical Overview of DNA - I

• In 1953, James Watson and Francis Crick
  introduced an elegant double-helical model for
  the structure of deoxyribonucleic acid, or DNA
• When T. H. Morgans group (1907) showed that
  genes are located on chromosomes, the two
  components of chromosomesDNA and proteinbecame
  candidates for the genetic material

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Historical Overview of DNA - II

• Frederick Griffith (1928) mixed heat-killed
  bacterial remains of a pathogenic strain with
  living cells of a 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
• Oswald Avery, Maclyn McCarty, and Colin MacLeod
  (1944) announced that the transforming substance
  was DNA

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Historical Overview of DNA - III

• More evidence for DNA as the genetic material
  came from studies of viruses that infect bacteria
• Such viruses, called bacteriophages (or phages),
  are widely used in molecular genetics research
• In 1952, Alfred Hershey and Martha Chase
  performed experiments showing that DNA is the
  genetic material of a virus (bacteriophage or
  phage) that infects bacteria known as T2 (phage)

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Additional Evidence That DNA Is the Genetic
Material
Sugarphosphate backbone
5? end
Nitrogenous bases
Thymine (T)

• It was known that DNA is a polymer of
  nucleotides, each consisting of a nitrogenous
  base, a sugar, and a phosphate group

Adenine (A)
Cytosine (C)
DNA nucleotide
Phosphate
Sugar (deoxyribose) 3? end
Guanine (G)
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• In 1950, Erwin Chargaff 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

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Building a Structural Model of DNA

• Rosalind Franklin used a technique called X-ray
  crystallography to study molecular structure
• Franklin produced a picture of the DNA molecule
  using this technique

Franklins X-ray diffraction photograph of DNA
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• Franklins X-ray crystallographic images of DNA
  enabled Watson to deduce that DNA was helical
• Franklin concluded that there were two
  antiparallel sugar-phosphate backbones, with the
  nitrogenous bases paired in the molecules
  interior
• In 1953, James Watson and Francis Crick
  introduced an elegant double-helical model for
  the structure of deoxyribonucleic acid, or DNA
• The X-ray images also enabled Watson to deduce
  the width of the helix and the spacing of the
  nitrogenous bases
• The width suggested that the DNA molecule was
  made up of two strands, forming a double helix

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5? end
Hydrogen bond
3? end
1 nm
3.4 nm
3? end
0.34 nm
5? end
(c) Space-filling model
(b) Partial chemical structure
(a) Key features of DNA structure
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• Watson and Crick thought the like bases paired
  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

Purine purine too wide
Pyrimidine pyrimidine too narrow
Purine pyrimidine width consistent with X-ray
data
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Pyrimidine
Purine

• They determined that adenine (A) paired only with
  thymine (T), and guanine (G) paired only with
  cytosine (C)
• The Watson-Crick model explains Chargaffs rules
  in any organism the amount of A T, and the
  amount of G C

Adenine (A)
Thymine (T)
Cytosine (C)
Guanine (G)
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Base Pairing to a Template Strand

• The two strands of DNA are complementary, each
  strand acts as a template
• In DNA replication,parent molecule unwinds, two
  new daughter strands are built

A
A
T
T
A
T
T
A
C
C
G
G
G
C
G
C
A
T
A
A
T
A
T
T
T
T
A
T
T
A
A
A
C
C
G
C
C
G
G
G
(c) Daughter DNA molecules, each consisting of
one parental strand and one new strand
(b) Separation of strands
(a) Parent molecule
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First replication
Second replication
Parent cell

• Conservative model
• the two parent strands rejoin

(b) Semiconserva- tive model one old strand and
one newly made strand
(c) Dispersive model each strand is a mix of old
and new
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DNA Replication Getting Started

• Replication begins at special sites called
  origins of replication - two DNA strands are
  separated, opening up a replication bubble
• A eukaryotic chromosome may have hundreds or even
  thousands of origins of replication
• Replication proceeds in both directions from each
  origin, until the entire molecule is copied

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Fig. 16-12
Origin of replication
Parental (template) strand
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
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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• Replication fork, a Y-shaped region where new DNA
  strands are elongating

Primase primase starts an RNA chain from scratch
and adds RNA nucleotides one at a time using the
parental DNA as a template
Single-strand binding proteins bind to and
stabilize single-stranded DNA until it can be
used as a template
Topoisomerase corrects overwinding ahead of
replication forks by breaking, swiveling, and
rejoining DNA strands
3?
5?
3?
RNA primer The primer is short (510 nucleotides
long), and the 3? end serves as the starting
point for the new DNA strand
5?
5?
3?
Helicase untwists the double helix at the
replication forks
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Synthesizing a New DNA Strand

• Enzymes called DNA polymerases catalyze the
  elongation of new DNA at a replication fork
• Most DNA polymerases require a primer (short RNA
  strand)and a DNA template strand

• DNA polymerases cannot initiate synthesis of a
  polynucleotide they can only add nucleotides to
  the 3? end

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• Each nucleotide that is added to a growing DNA
  strand is a nucleoside triphosphate
• dATP supplies adenine to DNA and is similar to
  the ATP of energy metabolism
• The difference is in their sugars dATP has
  deoxyribose while ATP has ribose
• As each monomer of dATP joins the DNA strand, it
  loses two phosphate groups as a molecule of
  pyrophosphate

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New strand 5? end
Template strand 3? end
5? end
3? end
Sugar
T
A
A
T
Base
Phosphate
C
G
G
C
G
G
C
C
DNA polymerase
3? end
A
A
T
T
3? end
C
C
Pyrophosphate
Nucleoside triphosphate
5? end
5? end
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Antiparallel Elongation

• The double helix is antiparallel
• 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

• Along one template strand of DNA, the DNA
  polymerase synthesizes a leading strand
  continuously, moving toward the replication fork

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Overview
Origin of replication
Leading strand
Lagging strand
Primer
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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Overview
Origin of replication
Lagging strand
Leading strand

• 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

Lagging strand
2
1
Leading strand
Overall directions of replication
5?
3?
3?
5?
Template strand
RNA primer
3?
5?
3?
1
5?
3?
Okazaki fragment
5?
3?
1
5?
5?
3?
3?
2
5?
1
5?
3?
3?
5?
1
2
5?
3?
3?
5?
1
2
Overall direction of replication
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Fig. 16-17
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 Complex

• The proteins that participate in DNA replication
  form a large complex, a DNA replication machine
• The 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 and Repairing DNA

• DNA polymerases proofread - replacing any
  incorrect nucleotides
• In mismatch repair of DNA, repair enzymes correct
  errors in base pairing
• In nucleotide excision repair, a nuclease cuts
  out and replaces damaged stretches of DNA

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Replicating the Ends of DNA Molecules

• 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

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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
• An enzyme called telomerase catalyzes the
  lengthening of telomeres in germ cells

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A chromosome consists of a DNA molecule packed
together with proteins

• Chromatin - a complex of DNA and protein, found
  in the nucleus of eukaryotic cells
• Histones - 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)

• Chromatin is organized into fibers
• 10-nm fiber
• DNA winds around histones to form nucleosome
  beads
• Nucleosomes are strung together like beads on a
  string by linker DNA
• 30-nm fiber
• Interactions between nucleosomes cause the thin
  fiber to coil or fold into this thicker fiber

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Chromatid (700 nm)
30-nm fiber
Loops
Scaffold
30-nm fiber
300-nm fiber
Replicated chromosome (1,400 nm)
Looped domains (300-nm fiber)
Metaphase chromosome

• 30-nm fiber- nucleosomes interactions cause thin
  fiber to coil
• 300-nm fiber looped domains that attach to
  proteins
• Metaphase chromosome looped domains coil further

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• Most chromatin is loosely packed in the nucleus
  during interphase and condenses prior to mitosis
• Loosely packed chromatin is called euchromatin
• During interphase a few regions of chromatin
  (centromeres and telomeres) are highly condensed
  into heterochromatin
• Dense packing of the heterochromatin makes it
  difficult for the cell to express genetic
  information coded in these regions