Download Lecture 6 DNA structure replication DNA structure, replication, and

Genetics 2011
O tli off Ch
Outline
Chapter
t 6
• Ho
How investigators
in estigators pinpointed DNA as the
genetic material
• The elegant Watson
Watson-Crick
Crick model of DNA
structure
• How DNA structure provides for the storage
of genetic information
• How DNA structure gives rise to the
semiconservative model of molecular
replication
• How DNA structure promotes the
recombination of genetic information
Lecture 6
DNA structure,
structure replication
replication,
and recombination
ᯘ⩶ె ⪁ᖌ
http://lms.ls.ntou.edu.tw/course/136
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The chemical
composition of DNA
Chemical characterization localizes
DNA in the chromosomes.
• 1869 – Friedrich Meischer extracted a
weakly
y acidic,, p
phosphorous
p
rich material
from nuclei of human white blood cells
which he named nuclein.
nuclein
• DNA – deoxyribonucleic acid
DNA contains four kinds of
nucleotides linked in a
long chain
• Deoxyribose – a sugar; acidic
• Four subunits belonging
g g to class of
compounds called nucleotides linked together
by
y phosphodiester
p
p
bonds
•Lectured
Chromosomes
are composed of DNA.
by Han-Jia Lin
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Phosphodiester bonds –
covalent bonds joining
adjacent nucleotides
Polymer – linked chain of
subunits
b it
Fig. 6.2Lectured by Han-Jia Lin
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Are genes composed of DNA or
protein?
Bacterial transformation implicates
DNA as the substance of genes.
• DNA
• 1928 – Frederick Griffith – experiments
with
ith smooth
th (S),
(S) virulent
i l t strain
t i
Streptococcus pneumoniae, and rough
(R) nonvirulent
(R),
i l t strain
t i
• Only four different subunits make up DNA
DNA.
• Chromosomes contain less DNA than
protein
t i b
by weight.
i ht
• Bacterial transformation demonstrates
transfer of genetic material.
• Protein
• 20 different subunits – greater potential
variety of combinations
• Chromosomes contain more protein than
DNA by weight.
weight
• 1944 – Oswald Avery,
y Colin MacLeod,
and Maclyn McCarty determined that
DNA is the transformation material.
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G iffith experiment
Griffith
i
t
G iffith experiment
Griffith
i
t
Rough
colony
Smooth
colony
Fi 6
Fig.
6.3
3
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Fig. 6.3a
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Fig. 6.3 b
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A
Avery,
M
MacLeod,
L d M
McCarty
C t E
Experiment
i
t
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H
Hershey
h and
d Chase
Ch
experiments
i
t
• 1952 – Alfred Hershey and Martha
Chase provide convincing evidence that
genetic material.
DNA is g
• Waring blender experiment using T2
bacteriophage and bacteria
• Radioactive labels 32P for DNA and 35S
for protein
Fig. 6.4 a
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Hershey and Chase Waring blender
experiment
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Hershey and Chase Waring blender
experiment
Fi 6.5
Fig.
6 5 a,b
b
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Fig.
6.5 c
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The Watson
Watson-Crick
Crick Model: DNA is a
double helix.
• 1951 – James Watson learns about x-ray
diff ti pattern
diffraction
tt
projected
j t db
by DNA
• Knowledge of the chemical structure of
nucleotides (deoxyribose sugar, phosphate,
and nitrogenous base)
• Erwin Chargaff’s experiments demonstrate
that ratios of A and T are 1:1,, and G and C
are 1:1.
• 1953 – James Watson and Francis Crick
propose their double helix model of DNA
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structure
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X-ray
X
ray diffraction patterns produced by DNA fibers –
Rosalind Franklin and Maurice Wilkins
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DNA’ chemical
DNA’s
h i l constituents
tit
t
Deoxyribose
Phosphate
DNA’ chemical
DNA’s
h i l constituents
tit
t
Four nitrogenous bases
Purines
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Attachment off
base to sugar
Pyrimidine
Nucleoside
P i
Purine
nucleotide
Pyrimidine
P
i idi
nucleotide
Fig. 6.9b
Fig. 6.9a
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Fig. Lectured
6.7a by Han-Jia Lin
Addition of phosphate to nucleoside
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Fig. Lectured
6.7b by Han-Jia Lin
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A detailed look at
DNA
DNA䇻䇻s chemical
constituents
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Ch
Chargaff’s
ff’ ratios
ti
• In all organisms,
g
, ratios of A to T and G to C are
roughly 1:1
Nucleotides linked in a
directional chain
Phosphodiester bonds
always form covalent link
between 3' carbon of one
nucleoside and 5'
5 carbon
of the next nucleoside
Note the 5
5'-to-3
to 3' polarity
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Fig. 6.7c
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Complementary base pairing by
f
formation
ti
off h
hydrogen
d
b
bonds
d explain
l i
Chargaff’s
g
ratios.
The double helix structure of DNA
• DNA is double helix
• Strands are antiparallel
with a sugar-phosphate
backbone on outside and
pairs of bases in the
middle.
• Two
T o strands wrap
rap around
aro nd
each other every 30
Angstroms,
g
, once every
y 10
base pairs.
• Two chains are held
t
together
th by
b h
hydrogen
d
bonds between A-T and
G-C base p
pairs.
• Base pairs consist of
h d
hydrogen
b
bonds
d ((weak
k
electrostatic bonds)
between a purine and a
pyrimidine (G with C, A
with T)
• Consistent with Chargaff's
rules
l
• Each base p
pair has ~
same shape
Fig. 6.8
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Fig. Lectured
6.9 by Han-Jia Lin
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Double helix may assume
alternative forms.
• Structurally, purines (A and G) pair
best with pyrimadines (T and C).
Thus A pairs with T and G pairs
• Thus,
with C, also explaining Chargaff’s
ratios.
ratios
B form DNA forms rightB-form
right
handed helix and has a
smooth backbone
Fig. 6.12
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Prokaryotes
Mitochondria
Chloroplasts
Vi
Viruses
• Some viruses carry
y single-stranded
g
DNA.
• 1.
1 bacteriophages
• Some viruses carry RNA.
• 1. e.g., AIDS
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Four q
questions about how DNA structure
relates to genetic functions
• S
Some DNA molecules
l
l are circular
i l
instead of linear.
1.
2
2.
3.
4
4.
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•
•
•
•
Z form DNA forms leftZ-form
left
handed helix and has an
irregular backbone
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How does the molecule carry information?
• Base sequence
How is that information is copied for transmission to
future generations?
• DNA replication
li ti
What mechanisms allow the genetic information to
change?
• Recombination
• Mutations
M t ti
((chapter
h t 7)
How does DNA-encoded information govern the
expression of phenotype?
• Gene functions (chapter 8)
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DNA stores information in the sequence
of its bases.
Some viruses use RNA as the
repository of genetic information.
(a) Most genetic information
is "read" from unwound
DNA
e.g. synthesis of DNA or
RNA
(b) Some
S
genetic
ti iinformation
f
ti
is accessible within doublestranded
st
a ded DNA
e.g. DNA-binding proteins that
regulate gene expression
Fig. 6.14
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Fig. 6.13
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DNA replication: Copying genetic
information for transmission to the next
g
generation
• Complementary base pairing produces
semiconservative replication.
• Double helix unwinds
• Each strand acts as template
• Complementary base pairing ensures that
T signals addition of A on new strand, and
G signals addition of C
C.
• Two daughter helices produced after
replication
li i
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Fig. 6.14
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Experimental proof of semiconservative replication –
three possible models
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Meselson Stahl experiments confirm
Meselson-Stahl
semiconservative replication.
• Semiconservative
replication – Watson and
Crick model
• Conservative replication:
parental double helix
The p
remains intact; both strands
of the daughter double helix
are newly synthesized
synthesized.
• Dispersive replication: At
completion,
p
, both strands of
both double helices contain
both original and newly
synthesized material
material.
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DNA synthesis
proceeds in a 5' to
3' direction
3
Th mechanism
The
h i
off DNA replication
li ti
• Arthur Kornbuerg, a nobel prize winner
and other biochemists deduced steps of
replication.
Template and newly
synthesized strands are
antiparallel
• Initiation
I iti ti
• Proteins bind to DNA and open up double helix.
• Prepare DNA for complementary base pairing
• Elongation
• Proteins connect the correct sequences of
nucleotides into a continuous new strand of
DNA
DNA.
DNA polymerase adds nucleotides
to 3'-OH of the new strand
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Fig. 6.19
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The mechanism of DNA replication:
Initiation
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The mechanism of DNA replication:
Initiation (cont)
• Preparation of double helix for complementary base
pairing
• Single-strand binding proteins keep the DNA helix
open
• Primase synthesizes RNA primer
• Primers are complementary and antiparallel to each
t
template
l t strand
t d
Initiation begins at
the origin (Ori) of
replication
Initiator protein binds
to Ori
Helicase unwinds the
helix
Two replication forks
are formed
Feature Fig.
g 6.20a
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The mechanism of DNA replication:
Elongation
The correct nucleotide sequence is copied from
template strand to newly synthesized strand
of DNA
DNA polymerase III catalyzes phosphodiester
bond formation between adjacent nucleotides
(polymerization)
Feature Fig. 6.20a (cont)
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The mechanism of DNA replication:
Elongation (cont)
• Leading strand has continuous synthesis
• Lagging strand has discontinuous synthesis
• Okazaki fragment – short DNA fragments on
lagging strand
Feature Fig. 6.20b
(cont)
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Feature Fig. 6.20b
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The mechanism of DNA replication:
Elongation (cont)
• DNA polymerase I replaces RNA primer
with DNA sequence
• DNA ligase
g
covalently
y jjoins successive
Okazaki fragments together
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E
Enzymes
involved
i
l d in
i replication
li ti
• Pol III – produces new stands of
complementary DNA
• Pol I – fills in gaps between newly
synthesized Okazaki segments
• DNA helicase – unwinds double helix
• Single-stranded binding proteins – keep helix
open
• Primase – creates RNA primers to initiate
synthesis
• Ligase
Li
– welds
ld ttogether
th Ok
Okazaki
ki ffragments
t
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Feature Fig. 6.20b (cont)
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The bidirectional replication of a circular
bacterial chromosome: An overview
The bidirectional
replication of a circular
bacterial chromosome:
An overview (cont)
DNA topoisomerases relax
supercoils by cutting the
sugar phosphate
backbone bonds
strands of DNA
• Replication proceeds
in two directions from
a single
i l O
Orii
• Unwinding of DNA
creates supercoiled
DNA ahead of
replication fork
Unwound
U
db
broken
k strands
t d
then sealed by ligase
Synthesis continues
bidirectionally until
replication
li i fforks
k meet
Fig. 6.21a, b
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Fig. 6.21c-f
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Cells must ensure accuracy of genetic
information.
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Recombination reshuffles the
information content of DNA.
• During recombination, DNA molecules
b k and
break
d rejoin.
j i
g - Experimental
p
• Meselson and Weigle
evidence from viral DNA and radioactive
p
isotopes
• Coinfected E. coli with light and heavy
strains of virus after allowing time for
recombination
• Separated on a CsCl density gradient
Three ways to ensure fidelity of DNA
information
Redundancy
•Redundancy
• Basis for repair of errors that occur during
replication or during storage
•Enzymes repair chemical damage to
DNA.
Errors during replication are rare
rare.
•Errors
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Heteroduplexes mark the spot of
recombination.
DNA molecules break and rejoin during
recombination: The experimental evidence
• M. Meselson and J. Weigle, co-infected E. coli with
radio-labeled phage
• Bacteriophage lambda with genetic markers grown
on E. coli in media with heavy (13C and 15N) or light
(12C and
d 14N) isotopes
i
Separated phage DNA on
CsCl density gradient
Genetic recombinants had
DNA with hybrid densities
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• Products of recombination are
always in exact register; not a
single base pair is lost or gained.
• Two strands do not break and
rejoin at the same location; often
they are hundreds of base pairs
apart.
apart
• Region between break points is
called heteroduplex.
heteroduplex
• One strand is maternal and
other
ot
e iss paternal
pate a
• Strands can have mismatches
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Mismatches in
h t
heteroduplexes
d l
can b
be
repaired
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Experimental observations that led to
development of a model of recombination
• DNA repair enzymes
eliminate mismatches
• Tetrad analysis in yeast showed that only two of
the four chromatids are recombinant
• Either allele can be converted
• Recombination occurs only between homologous
regions and is highly accurate
• Gene conversion – deviations
from expected 2:2
segregation e
segregation,
e.g.
g 3:1 or 1:3
• Crossover sites often associated with
heteroduplex regions
• In yeast, gene conversion
occurs 50:50
50 50 with
ith and
d without
ith t
crossing over of flanking
markers
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Fig. 6.23c
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• Gene conversion can occur in absence of
crossing over
• Not all recombination leads to crossovers
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Double stranded break model of
meiotic recombination
• Homologs physically break
break, exchange parts
parts, and
rejoin.
• Breakage
g and repair
p create reciprocal
p
p
products
of recombination.
• Recombination events can occur anywhere
along
l
the
th DNA molecule.
l
l
• Precision in the exchange prevents mutations
from occurring during the process
process.
• Gene conversion can give rise to unequal yield
of two different alleles
alleles. 50% of gene
conversions are associated with crossing over of
adjacent chromosomal regions, and 50% of
gene conversions
i
are nott associated
i t d with
ith
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crossing over.
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Double-strand
Doublestrand--break repair model
of meiotic recombination
• Homologous chromosomes break, exchange DNA,
and rejoin
j
• Breakage and repair creates reciprocal products of
recombination
• Recombination events can occur anywhere along the
DNA
• Precision in the exchange
g ((no g
gain or loss of
nucleotide pairs) prevents mutations from occurring
• Gene conversion can give rise to an unequal yield of
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two different alleles
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Step 1 in the model of recombination:
Double--strand break formation
Double
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Step 2 in the model of recombination:
Resection
Dmc1
D
1 breaks
b k phosphodiester
h
h di t b
bonds
d off b
both
th strands
t d off
one chromatid
ƒ Spo11 in yeast is homologous to Dmc1 of
multicellular eukaryotes
• 5' ends of each broken strand are
degraded to create 3䇻
3 single
single-stranded
stranded
tails
Feature
Fig. 6.24
Feature Fig. 6.24 (cont)
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Step 3 in the model of recombination:
First strand invasion
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Step 4 in the model of recombination:
Formation of double Holliday junctions
• One single-strand tail invades a nonsister chromatid and forms stable
heteroduplex
• Displacement
Di l
t lloop (D
(D-loop)
l
) ffrom
invaded chromatid is stabilized by
single-strand binding protein
• D-loop enlarged by new DNA synthesis at 3'end of invading
g strand
• New DNA synthesis fills in gap in bottom
strand using displaced strand as template
Feature Fig. 6.24 (cont)
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Feature Fig. 6.24 (cont)
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Step 5 in the model of recombination:
Branch migration
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Step 6 in the model of recombination:
The Holliday intermediate
• Heteroduplex region of both DNA
molecules is lengthened
Feature
Fig. 6.24
(cont)
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Feature Fig. 6.24 (cont)
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Step 7 in the model of recombination:
Alternative resolutions
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Step 7 in the model of recombination:
Alternative resolutions
• Cutting of Holliday junctions by endonucleases in
either vertical or horizontal plane is equally likely
• Cutting of Holliday junctions by endonucleases is
q
y likelyy in either vertical or horizontal p
plane
equally
Feature
Fig. 6.24
(cont)
Feature
Fig. 6.24
(cont)
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Step 8 in the model of recombination:
Probability of crossover occurring
E
Essential
ti l Concepts
C
t
• N
Non-crossover occurs when
h b
both
th jjunctions
ti
are
resolved in same plane
• Crossover occurs with the two junctions are
resolved in different planes
Feature Fig. 6.24 (cont)
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• DNA is the nearly universal genetic material.
• The Watson-Crick model shows that DNA is a
double helix composed of two antiparallel
strands of nucleotides: each nucleotide
consists of one of four nitrogenous bases
((A,T,C,
, , , or G),
), a deoxyribose
y
sugar,
g , and a
phosphate. An A pairs with a T and a G pairs
with a C.
• DNA carries information in the sequence of its
bases which may follow one another in any
bases,
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order.
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