DNA Structure and Replication

1
 Nucleic Acids – polymers of nucleotides
Primary structure is the sequence of nucleotides attached to one another by
phosphodiester bonds
Nucleotide = base + sugar + phosphate
  DNA = deoxyribonucleic acid
Types of Nucleic Acids:
  - polymer of deoxyribonucleotides
   base + deoxyribose + phosphate
  RNA = ribonucleic acid
  - polymer of ribonucleotides
   base + ribose + phosphate

2
Different kinds of phosphate bonds

3
Nitrogenated bases are purines and pyrimidines

Cytosine can be converted to uracil by cytosine deaminase

4
 DNA – a polydeoxyribonucleotide
  Composed of monodeoxyribonucleotides covalently linked by 3’  5’
phosphodiester bonds

  Has polarity

  Bases always written in sequence from 5’end of chain on the left to the 3’
end on the right

5
Complementarity of the base pairs enable stacking in the
energetically most favorable condition in the double helix
The main glue for the double helix is the stacking of bases between adjacent bases

 The stacking of adjacent bases

stabilize single stranded DNA and
double stranded DNA

 In double stranded DNA the

separation between the hydrophobic
bases and the hydrophilic
components (sugar and phosphate)
is more pronounced than the single
stranded DNA thereby making more
energetically favorable

 Additional stability comes from

hydrogen bonds between the bases.
Indirectly they optimize, because of
their dependence in directionality,
the stacking of bases 6
 Chargaff’s rules of base composition
 The total amount of pyrimidine nucleotides (T +C) always equals the total
amount of purine nucleotides (A + G)

  The amount of T always equals the amount of A, and the amount of C

always equals the amount of G

7
 One polynucleotide chain of the DNA double helix is
always the complement of the other

  In any double-stranded DNA, A = T and G = C. Purines = pyrimidines

  Given the sequence of bases on 1 chain, the sequence of bases on the complementary chain
can be determined
  Tautomers of the bases give rise to configurational isomers whereby the hydrogen location 8
differs
Double-stranded DNA exists in 3 forms (A, B and Z)
B form is usually found under
physiologic conditions
DNA

RNA

9
Comparisons of DNA double-stranded forms

10
11
  Denaturation (Melting) of DNA
  Separation of the 2 DNA strands in the double helix
  Occurs when H-bonds between the paired bases are disrupted
  Occurs in the laboratory if solution is heated
  The higher the GC content, the higher the Tm, the temperature at
which ½ of the helical structure is lost

  Renaturation (Reannealing)
  Process by which complementary DNA strands reform the double helix 12
 Packaging of DNA Inside the Cell

  dsDNA

  Histones

  Nucleosomes

  Linker DNA

  Nucleofilament

  30 nm fiber

  Protein Scaffold

  Chromosome
13
 Because DNA molecules are so large, they require special
packaging to enable them to reside within cells
 E. coli: DNA has 4 x 106 base
pairs, 2 mm long
  Circular DNA is supercoiled

 Human: DNA contains a total of 4

x 109 base pairs
  DNA from all 46
chromosomes in a diploid
human cell, placed end to end,
would stretch about 2 m (> 6 ft)

  DNA has multiple levels of

packaging that is facilitated by
binding with histones 14
Intermediate filaments are like ropes made of long, twisted
strands of protein like the nuclear lamins

15
Nuclear lamins are fibrous proteins that form intermediates
filaments

16
 Histones – small proteins
  Positively charged at physiologic pH
 Result of high content of lysine
and arginine
 Form ionic bonds with
negatively charged DNA
 Help neutralize the negatively
charged DNA phosphate groups
 They contain an N-terminal tail
and a globular histone fold (at
least three alpha helices
connected by short loops)

  With 5 classes: H1 – associated with

linker DNA
H2A
H2B Form the core of a
H3 nucleosome
H4
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 DNA Packaging
  DNA double helix are wound nearly twice
around a core of histones
(2 each of H2A, H2B, H3 and H4)=
nucleosome

  The histone H3-H4 dimer and H2A-

H2B dimer are formed from the
handshake interaction

  An H3-H4 tetramer forms and binds

to the DNA

  Two H2A-H2B dimers are then

added, to complete the nucleosome

  Note that all eight N-terminal tails of

the histones protrude from the disc-
shape core structure

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Nucleosomes are joined by “linker” DNA (Nucleofilaments)
  corresponds to an extended polynucleosome chain
  has the “beads-on-a-string” appearance
  H1 – tissue-specific and species-specific of histones
facilitates the packing of nucleosomes into more
compact structures

Linker DNA: about 50 base pairs long associated with H1

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 Chromatin packing
represents a compaction of 10,000 fold

Chromosome structure found

in interphase

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22
  Semi-conservative
 half of the parental DNA molecule (1 strand)

 conserved in each new double helix

  paired with a newly synthesized
complementary strand

  Exhibits polarity

23
 The DNA replication fork is asymmetrical
 DNA polymerases
–  Responsible for copying the DNA templates
–  Synthesize the new DNA strands in the 5’  3’ direction
–  The parental nucleotide strand is read in the 3’  5’ direction

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 Replication in prokaryotes
  Separation of the 2 complementary DNA strands (helicase)
  Single-stranded DNA binding proteins
  Topoisomerases

  Primer synthesis (an RNA primer required)

  DNA synthesis
  DNA polymerase III (prokaryotes)
  Okazaki fragments on lagging strand
  Primers removed by DNA polymerase I, which fills in gaps
  DNA Ligase

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Replication in Prokaryotes
 DNA helicases Special proteins help to open up the
  move into neighboring DNA double helix in front of the
double-stranded region, forcing
the strands apart, in effect unwinding the helix replication fork

 When strands separate, SSB proteins bind,

preventing reformation of the double helix

 Single-stranded DNA-binding (SSB) proteins

(helix destabilizing proteins) bind to single-
stranded DNA
 SSB proteins
  keep the 2 strands of DNA separated in the area of the
replication origin
  also protect the DNA from nucleases that cleave
single-stranded DNA

26
 Replication in Prokaryotes
Separation of the 2 Complementary DNA strands
Primer synthesis
 Primase – DNA-dependent RNA polymerase
 Synthesizes short stretches of RNA (about 10 nucleotides
long) that are complementary and anti-parallel to the DNA
template

 DNA polymerases require an RNA primer with a free

hydroxyl group on the 3’-end of the RNA strand
  Hydroxyl group serves as the first acceptor of a nucleotide
by action of DNA polymerase 27
Replication in Prokaryotes
Separation of the 2 Complementary DNA strands
Primer Synthesis
DNA synthesis

28
 Replication in Prokaryotes
DNA synthesis
  DNA Polymerase III
 The chemistry of DNA replication, repair and recombination is largely that of the
phosphodiester bonds that link neighboring nucleotides in a DNA chain

  Has 5’  3’ polymerase activity

  Uses 5’-deoxyribonucleotide triphosphates

  All 4 deoxyribonucleotide triphosphates must be present (dATP, dTTP, dCTP, dGTP)

  DNA synthesis stops if (when) a nucleotide is depleted
 The elongation of the 3OH end involves a transesterification reaction

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Initiation of DNA replication

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 Replication in prokaryotes

  Separation of the 2 Complementary DNA strands

  Leads to the formation of a “V” where active DNA synthesis occurs
  The replication fork moves along the DNA molecule as synthesis occurs
using a polymerase dimer

31
DNA synthesis on the leading and lagging strands

32
33
 The chemistry of DNA synthesis
 The addition of a deoxyribnucleotide to
the 3’ end of a polynucleotide chain (the
primer strand) is the fundamental reaction
by which DNA is synthesized

 Base-pairing between an incoming

deoxyribonucleotide triphosphate and an
existing strand of DNA (template strand)
guides the formation of DNA and causes it
to have a complementary nucleotide
sequence

 The shape of DNA polymerase resembles

a right hand in which the palm, fingers,
and the thumb grasp the DNA and form
the active site

34
 The high fidelity of DNA replication requires several
proofreading mechanisms

 The fidelity of copying DNA during replication is such that only about 1 mistake occurs for
every 109 nucleotides copied

 If the DNA polymerase did nothing special when a mispairing occurred between an
incoming deoxyribonucleotides triphosphate and the DNA template, the wrong nucleotide
would be incorporated in the DNA chain, producing frequent mutations

 The high fidelity of DNA replication, however, depends not only in the initial-base pairing
but also on several “proofreading” mechanisms that act sequentially to correct any initial
mispairing that might have occurred.

 DNA polymerase performs the first proofreading step just before a new nucleotide is added
to the growing chain 35
 Only DNA replication in the 5’ to 3’ direction allows
efficient error correction
 The need for accuracy probably explains
why DNA replication occurs only in the 5’ to
3’ direction

 If there were a DNA polymerase that

added deoxyribonucleoside triphophate in
the 3’ to 5’ direction, the growing 5’-chain
end, rather than the incoming
mononucleotide, would provide the
activating triphosphate needed for the
covalent linkage

 In this case, the mistakes in polymerization

could not be simply hydrolyzed away,
because the bare 5’-chain end thus created
would immediately terminate DNA synthesis

 It is therefore possible to correct

mismatched base only if it has been added to
the 3’ end of a DNA chain 36
37
3’ 5’

5’ 3’

3’ 5’

5’
3’

38
Replication in Prokaryotes
DNA synthesis

  DNA Polymerase III

- has 5’  3’ polymerase activity
3’ 5’

A G G T C C G G A T
T C!  Adds deoxyribonucleotides one at a
time to the 3’ end of the growing chain
Replication
5’  Sequence of nucleotides added is
dictated by the base sequence of the
template strand with which the incoming
nucleotides are paired

DNA Polymerase III

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Replication in Prokaryotes
DNA synthesis

 DNA Polymerase III

 has 5’  3’ polymerase activity

3’ 5’

A G G T C C G G A T
T C! C!
5’

40
Replication in Prokaryotes
DNA synthesis

  DNA Polymerase III

 has proofreading activity

3’ 5’

A G G T C C G G A T
T C C G
5’

DNA polymerase III recognizes its mistake

41
Replication in Prokaryotes
DNA synthesis

  DNA Polymerase III has proofreading activity

3’ 5’

A G G T C C G G A T
T C C
5’ DNA polymerase III uses its
3’  5’ exonuclease activity to backup

42
Replication in Prokaryotes
DNA synthesis

  DNA Polymerase III

- has proofreading activity

3’ 5’

A G G T C C G G A T
T C C A
5’
DNA polymerase III repairs the mismatch.

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Replication in Prokaryotes
DNA synthesis

 DNA Polymerase III

 has proofreading activity

3’ 5’

A G G T C C G G A T
T C C A G
5’ DNA polymerase III continues
with its 5’  3’ activity.

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 Replication in Prokaryotes
DNA synthesis

3’ 5’

A G G T C C G G A T
T C C C C U A
5’

RNA Primer

Newly synthesized DNA

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 Replication in Prokaryotes DNA synthesis

  DNA Polymerase III

3’ 5’

A G G T C C G G A T
T C C A G G C C U A
5’

Once DNA polymerase III reaches the RNA primer, it can

proceed no further

46
Replication in Prokaryotes
DNA synthesis

 DNA Polymerase III

3’ 5’

A G G T C C G G A T
T C C A C C U A
5’

47
 Replication in Prokaryotes
DNA synthesis

  DNA Polymerase III

3’ 5’

A G G T C C G G A T
T C C A G C C U A
5’

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 Replication in Prokaryotes
DNA synthesis

  DNA Polymerase I

3’ 5’

A G G T C C G G A T
T C C A G G C C U A
5’

DNA polymerase I takes its

DNA polymerase III dissociates place
from the DNA strand.

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 Replication in Prokaryotes
DNA synthesis

  DNA Polymerase I

3’ 5’

A G G T C C G G A T
T C C A G G C C U A
5’
DNA polymerase I has:
  5’ to 3’ exonuclease activity
 Able to remove the RNA primer

  5’ to 3’ polymerase activity
 Replaces the removed RNA primer with deoxyribonucleotides

  3’ to 5’ exonuclease proofreading activity 50

 Replication in Prokaryotes
DNA synthesis

  DNA Polymerase I

3’ 5’

A G G T C C G G A T
T C C A G G C C U A
5’

DNA polymerase I has:

  3’ to 5’ exonuclease activity
  “proofreads” the newly synthesized DNA chain

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 Replication in Prokaryotes
DNA synthesis

  DNA Polymerase I

3’ 5’

A G G T C C G G A T
T C C A G G C C T A
5’

 removal/synthesis/proofreading continues
one nucleotide at a time until the RNA is totally
degraded and the gap is filled with DNA

52
 Replication in Prokaryotes
DNA synthesis

  DNA Polymerase I

3’ 5’

A G G T C C G G A T
T C C A G G C C T A
5’

Nick in the phosphodiester backbone

C

U 53
 Replication in Prokaryotes
DNA synthesis

  DNA ligase

3’ 5’

A G G T C C G G A T
T C C A G G C C T A
5’

DNA ligase seals the nick

C

U
54
 Replication in Prokaryotes

DNA synthesis

  DNA ligase

3’ 5’

A G G T C C G G A T
T C C A G G C C T A
5’

A
C

55
56
 Summary of key components of DNA
replication in prokaryotes on the lagging
strand

57
Replication in Prokaryotes
DNA synthesis DNA ligase
  Catalyzes the final phosphodiester linkage between:
a. 5’ phosphate group on the DNA chain synthesized by DNA polymerase III and
b. 3’ hydroxyl group on the chain made by DNA polymerase I

High E

HE
Is kept

58
Comparison of exonucleases and endonucleases

59
DNA replication in eukaryotes occurs in the S phase

60
 DNA replication in eukaryotes (2)
  Primase activity of polymerase α/primase
complex initiates synthesis of an RNA primer

  After RNA primer reaches 10 nucleotides, the

polymerase α 5’  3’ polymerase activity takes
over
  Adds about 20-30 deoxyribonucleotides

  Polymerase α/primase dissociates

  Polymerase δ complex binds and elongates the

chain.

  As the replication complex approaches an earlier

RNA primer, the primer is degraded by RNAase
H and FEN 1

  Gap is filled by polymerse δ continued elongation

of the Okazaki fragment

  Remaining nick is sealed by DNA ligase 61

 Replication of DNA:origins and replication forks
A stretch of DNA that is rich in As and Ts and thereby easy to unwind
A) Small prokaryotic circular DNA contains only one origin of replication

B) Very long eukaryotic DNA contains from 400 to 10000 orings of replication

62
 Eukaryotic DNA replication
  Similar to that in prokaryotes

  Daunorubicin and doxorubin are used as in the treatment of leukemias.

  They interfere with the activity of topoisomerase II by intercalating between

bases of DNA. This prevent DNA replication in tumor cells

  Differences:
  With multiple origins of replication
  Centromeres (they allow separation of chromosomes into each daughter cell)
  Linear DNA
  Telomerase is required to maintain the length of the chromosome
  RNA primers are removed by RNAse H
  The primer is a mixture of RNA and DNA
  RNAase H and Fen degrades the primer
  Eukaryotic polymerases
•  DNA polymerase α and δ : lagging strand synthesis
•  DNA polymerase δ : leading strand synthesis (α initiates)
•  DNA polymerase γ: mitochondrial DNA synthesis
•  DNA polymerase β and ε: DNA repair 63
64
Types of DNA polymerases

65
Replication in Prokaryotes

DNA topoisomerases add or remove supercoils in the helix

  DNA topoisomerases alter the linking number of DNA
  With both nuclease (strand-cutting) and ligase (strand-resealing) activities
  With 2 types:
  Type I DNA topoisomerase
  Type II DNA topoisomerase

66
 Topoisomers are DNA molecules differing only in
linking number
A
D

Lk = Number of nucleotides
10.5

B
L=T+W

L=linking number (defined as the

C number of times one strand of DNA
winds around the other)

T= turns

W= writhing number refers to the number

of super-helix turns
67
Replication in Prokaryotes

DNA topoisomerases remove supercoils in the helix

Type I topoisomerase
68
Topoisomerase I (heterotetramer of gyrA and gyr B)
mechanism involves phosphotyrosine intermediates

69
Replication in Prokaryotes

DNA topoisomerases remove or add supercoils in the helix

Viral DNA

Topo II 5’ 30’ mins Type II topoisomerase

70
Replication in Prokaryotes
DNA gyrase
 A type II topoisomerase found in E. coli
 Able to introduce negative supercoils into relaxed circular
DNA using energy from the hydrolysis of ATP
 Facilitates the future replication of DNA because the (-) supercoils
neutralize the positive supercoils introduced during opening of the
double helix
 A swivel ahead of the replication machinery
 DNA gyrase is the target of many antibiotics.
 Topoisomerase inhibitors such as nalidixic acid freeze the covalent DNA-
protein links; nalidixic acid is used for urinary tract infections.
Ciprofloxacin is another inhibitor of topoisomerases which is used to
prevent and treat antrax

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Inhibitors of topoisomerases in eukaryotes and prokaryotes

Antibiotics

Anticancer drugs

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Reverse transcriptase

 Uses RNA as template for the

synthesis of DNA

 Present in some viruses

  In contrast to DNA polymerase,

RNA polymerase do not have 3’ 5
proofreading activity thereby is prone
to errors and high rate of mutations
 Example: human
immunodeficiency virus

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Production of the virus that causes AIDS

74
Only eukaryotes contain genomes in which some
genes have introns

75
The replication problem at chromosome ends
in linear chromosomes

76
 Telomeres
 Consist of many tandem repeats of a 6-nucleotide DNA sequence (TTGGGG
tetrahymena
 And human TTAGGG)

 Do not encode proteins

 Undergo cycles of shortening and addition of new repeats to the 3’ end by

telomerase

 Telomerase is active in germ cells, fetal cells and cancer cells

 Generally not active in adult somatic cell

 Generally not active in adult somatic cells

77
 Mechanism of action of telomerase

78
Telomerase has reverse transcriptase activity
Central dogma of molecular biology

80
List the forces that hold the DNA double helix together as a stable unit
-
Hydrogen bonding, dipole interaction

81
What is the polarity of the growing leading strand
synthesize by DNA polymerase III?

What is the polarity for the lagging strand?

What is the polarity of the template in both cases?

82
If species A has more DNA per nucleus than species B, does A necessarily
have more genes than B?

Please explain

83
DNA topoisomerases play important roles in DNA replication and supercoiling.
These enzymes are also the targets for certain anticancer drugs. Eric Nelson and
his colleagues studied m-AMSA, one of the anticancer compounds that acts on
topisomerase enzymes. They found that m-AMSA stabilizes an intermediate
produced in the course of the topoisomerase’s action.
The intermediate consisted of the topoisomerase bound to the broken ends of the
DNA. Breaks in DNA that are produced by anticancer compounds such as m
-AMSA inhibit the replication of the cellular DNA and thus stop cancer cells from
proliferating.

Propose a mechanism for how m-AMSA and other anticancer agents that target
topoisomerase enzymes taking part in replication might lead to DNA breaks and
chromosome rearrangments.

84
 Linking number

A closed-circular DNA molecule in its relaxed form has an Lk of 500. Approximately

how many base pairs are in this DNA? How is the linking number altered (increases,
decreases, doesn’t change, becomes undefined) when
(a) a protein complex is bound to form a nucleosome,
(b) one DNA strand is broken
(c) DNA gyrase and ATP are added to the DNA solution, or
(d) the double helix is denatured by heat?

85
 DNA replication

Kornberg and his colleagues incubated soluble extracts of E. coli with a mixture of
dATP, dTTP, dGTP, and dCTP, all labeled with 32P in the α-phosphate group. After a
time, the incubation mixture was treated with trichloroacetic acid, which precipitates
the DNA but not the nucleotide precursors. The precipitate was collected, and the
extent of precursor incorporation into DNA was determined from the amount of
radioactivity present in the precipitate.

(a)  If any one of the four nucleotide precursors were omitted from the incubation
mixture, would radioactivity be found in the precipitate? Explain.

(b) Would 32P be incorporated into the DNA if only dTTP were labeled? Explain.

(c) Would radioactivity be found in the precipitate if 32P labeled the β or γ phosphate
rather than the a phosphate of the deoxyribonucleotides? Explain.

86
DNA polymerases are not able to prime replication, yet primase and other
RNA polymerases can. Some geneticist have speculated that the inability of
DNA polymerase to prime replication is due to its proofreading function.
This hypothesis argues that proofreading is essential for faithful
transmission of genetic information and that, because DNA polymerases
have evolved the ability to proofread, the cannot prime DNA synthesis.
Explain why proofreading and priming functions in the same enzyme might
be incompatible?

87
A number of scientist who study ways to treat cancer have become
interested in telomerase. Why would they be interested in
telomerase? How might cancer-drug therapies that target
telomerase work?

88
The enzyme telomerase is part protein and part RNA. What would be
the most likely effect of a large deletion in the gene that encodes the
RNA part of telomerase? How would the function of telomerase be
affected?

89
 Function of DNA ligase

Some E. coli mutants contain defective DNA ligase. When these mutants are exposed to 3H
-labeled thymine and the DNA produced is sedimented on an alkaline sucrose density
gradient, two radioactive bands appear. One corresponds to a high molecular weight
fraction, the other to a low molecular weight fraction. Explain.

90
Suppose a future scientist explores a distant planet and discovers a novel form of double
stranded nucleic acid. When this nucleic acid is exposed to DNA polymerases from E.
coli, replication takes place continously on both strands. What conclusion can you make
about the structure of this novel nucleic acid?

91
 DNA topology

In the presence of a eukaryotic condensin and a type II topoisomerase, the Lk of a relaxed

closed-circular DNA molecule does not change. However, the DNA becomes highly knotted.

Formation of the knots requires breakage of the DNA, passage of a segment of DNA through the
break, and religation by the topoisomerase. Given that every reaction of the topoisomerase would
be expected to result in a change in linking number, how can Lk remain the same?

92
 The DNA below is replicated from left to right. Label the
templates for leading strand and lagging strand synthesis.

(5')ACTTCGGATCGTTAAGGCCGCTTTCTGT(3')
(3')TGAAGCCTAGCAATTCCGGCGAAAGACA(5')

93
A suitable substrate for DNA polymerase is shown below. Label the
primer and template, and indicate which end of each strand must be
3' or 5'.

To observe DNA synthesis on this substrate in vitro, what

additional reaction components must be added?

94
DNA synthesis on the lagging strand in E. coli is a complex process known to
involve several proteins. Initiation of a new chain is catalyzed by the enzyme (a)
_____________, and elongation is catalyzed by the enzyme (b)______________.

Synthesis is discontinuous, yielding short segments called (c) _______________,

which are eventually joined by the enzyme (d)______________, which requires
the cofactor (e)___________.

95
What will be the effect on DNA replication of mutations that destroyed each of
the following activities in DNA polymerase I?

a. 3’5’ exonuclease activity

b. 5’3’ exonuclease activity
c. 5’3’ polymerase activity

96