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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
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
7
One polynucleotide chain of the DNA double helix is
always the complement of the other
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
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)
18
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
19
Chromatin packing
represents a compaction of 10,000 fold
20
22
Semi-conservative
half of the parental DNA molecule (1 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
24
Replication in prokaryotes
Separation of the 2 complementary DNA strands (helicase)
Single-stranded DNA binding proteins
Topoisomerases
DNA synthesis
DNA polymerase III (prokaryotes)
Okazaki fragments on lagging strand
Primers removed by DNA polymerase I, which fills in gaps
DNA Ligase
25
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
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
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
29
Initiation of DNA replication
30
Replication in prokaryotes
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
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
5’ 3’
3’ 5’
5’
3’
38
Replication in Prokaryotes
DNA synthesis
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
39
Replication in Prokaryotes
DNA synthesis
3’ 5’
A G G T C C G G A T
T C! C!
5’
40
Replication in Prokaryotes
DNA synthesis
3’ 5’
A G G T C C G G A T
T C C G
5’
41
Replication in Prokaryotes
DNA synthesis
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
3’ 5’
A G G T C C G G A T
T C C A
5’
DNA polymerase III repairs the mismatch.
43
Replication in Prokaryotes
DNA synthesis
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.
44
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
3’ 5’
A G G T C C G G A T
T C C A G G C C U A
5’
46
Replication in Prokaryotes
DNA synthesis
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
3’ 5’
A G G T C C G G A T
T C C A G C C U A
5’
48
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’
49
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
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’
51
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’
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’
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
B) Very long eukaryotic DNA contains from 400 to 10000 orings of replication
62
Eukaryotic DNA replication
Similar to that in prokaryotes
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
66
Topoisomers are DNA molecules differing only in
linking number
A
D
Lk = Number of nucleotides
10.5
B
L=T+W
T= turns
Type I topoisomerase
68
Topoisomerase I (heterotetramer of gyrA and gyr B)
mechanism involves phosphotyrosine intermediates
69
Replication in Prokaryotes
Viral DNA
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
71
Inhibitors of topoisomerases in eukaryotes and prokaryotes
Antibiotics
Anticancer drugs
72
Reverse transcriptase
73
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)
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?
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
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
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'.
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)______________.
95
What will be the effect on DNA replication of mutations that destroyed each of
the following activities in DNA polymerase I?
96