LECTURE 6

Structure & Function of

Nucleic Acids
Biology, Campbell & Reece; 7th Edn. Ch 16, pp. 293-305, Ch 19, pp. 359-361

By

Dr Mohamed Abumaree
Molecular Reproductive Biology & Immunology
College of Medicine
King Saud bin Abdulaziz University for Health Science
Riyadh
2009

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 DNA Structure
 DNA: a polymer of
nucleotides: consists
of:
1. A nitrogenous bases:
A, T, G & C

1. A pentose sugar
(deoxyribose)

2. A phosphate group
2
 DNA is made up of 2
strands
 Sugar–phosphate
backbones outside DNA
 Hydrophobic bases in DNA
 Bases are paired in the
complementary strand:
 A with T & G with C

So, A amount = T amount &

G amount = C amount
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 Pairs of bases
are held together
by hydrogen
bonds

A forms 2
hydrogen bonds
with T
G forms 3
hydrogen bonds
with C
 DNA Replication
 DNA is doubled & distributed equally to 2 daughter
cells
 DNA replication is remarkable in its speed &
accuracy
 >12 enzymes & proteins participate in DNA
replication
 DNA replication is more known in bacteria than in
eukaryotes
 DNA replication is similar for prokaryotes &
eukaryotes
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 The parent molecule
has 2 complementary
strands of DNA 1

 Each base is paired by

hydrogen bonding

The first step in

DNA replication is 2
the break of bonds
and the separation of
the 2 DNA strands
 Each parental strand
determines the order of
nucleotides a long a 3
new complementary
strand

 Nucleotides form the

sugar-phosphate
backbones of new
strands
 Each daughter DNA 4
molecule consists of one
parental strand & one
new strand
 Replication Origins
 Bacterial chromosome (circular) has a
single replication origins (a stretch of a
specific DNA sequence) where proteins
recognize & attach to separate the 2
strands & open up a replication “bubble”

 DNA replication proceeds in both

directions until the entire molecule is
copied

 In contrast to a bacterial chromosome, a

eukaryotic chromosome have
100s/1000s of replication origins 8
 Replication begins at specific sites
where the two parental strands
separate & form replication bubbles 1

The bubbles expand laterally, as

DNA replication proceeds in both 2
directions
Eventually, the replication bubbles
fuse & synthesis of the daughter 3
strands is complete

 In eukaryotes, DNA replication begins at many sites

along the giant DNA molecule of each chromosome
 At each end of a replication bubble is a replication fork
(Y–shaped region) where the new strands of DNA are
elongating
 New DNA Strand Elongation
 DNA polymerases
catalyze the new
DNA elongation at a
replication fork
 DNA polymerase
adds nucleotides to
the growing end of
the new DNA strand

As each monomer joins the growing end of a DNA

strand, it loses 2 molecules of P–Pi , which is then
hydrolyzed to 2 molecules of Pi
 Antiparallel Elongation
 2 ends of a DNA
strand are different
 2 strands of DNA are
antiparallel
 So, 2 new strands
formed during DNA
replication must also be
antiparallel to their
template strands
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 DNA polymerases add nucleotides only to the free 3′
end of a growing DNA strand, never to the 5′ end
 So, a new DNA strand can elongate only in the 5′ →
3′ direction
 Along one template
strand, DNA
polymerase synthesize a
complementary strand
continuously by
elongating the new
DNA in the mandatory 5′
→ 3′ direction
 DNA polymerase continuously adds nucleotides to the
complementary strand (leading strand) as the fork
progresses
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 To elongate the other new
strand of DNA in the
mandatory 5′ → 3′
direction, DNA polymerase
must work along the other
template strand in the
direction away from the
replication fork. This
mechanism is called lagging
strand

 Synthesis of leading & lagging strands occur

concurrently & at the same rate
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 Lagging strand synthesis is slightly delayed relative
to leading strand synthesis, because each new
fragment cannot be started until enough template is
exposed at the replication fork

 The leading strand elongates continuously,

continuously the
lagging strand is synthesized as a series of segments

 Once a replication bubble opens far enough,

enough a DNA
polymerase attaches to the template of lagging strand &
moves away from the replication fork to synthesize a
short DNA segment
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 As the bubble grows, another segment of the lagging
strand can be made in a similar way

 These segments of the lagging strand are called

Okazaki fragments

 The fragments are about 1,000 to 2,000 nucleotides

long in E. coli & 100 to 200 nucleotides long in
eukaryotes

 DNA ligase joins the Okazaki fragments to form a

single new DNA strand
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 Priming DNA Synthesis
 DNA polymerases cannot initiate the synthesis of a
polynucleotide, but only add nucleotides to 3′ end of an
already existing nucleotide chain (primer), which
consist of DNA/RNA that can initiate the replication of
cellular DNA
 Primer is a short stretch of RNA with an available 3′
end
 Primase can start an RNA chain from scratch
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 Primase joins RNA nucleotides together one
at a time, making a primer complementary to
the template strand at the location where
initiation of the new DNA strand will occur

 Primers are generally 5 to 10 nucleotides long

 One primer is required for DNA DNA

polymerase to begin synthesizing the leading
strand
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 For synthesis of the
lagging strand,
strand each
Okazaki fragment must be
primed separately

 Another DNA
polymerase replaces the
RNA nucleotides of the
primers with DNA
versions,
versions adding them onto
the 3′ end of the adjacent
Okazaki fragment
(fragment 2)
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 DNA polymerase cannot
join the final nucleotide of
this replacement DNA
segment to the first DNA
nucleotide of the Okazaki
fragment whose primer
was just replaced (fragment
1)

 DNA ligase accomplishes

this task, joining the sugar–
phosphate backbones of all
the Okazaki fragments into
a continuous DNA strand 20
 Proteins Assisting DNA Replication
 Helicase untwists the double helix at the replication
forks,
forks separating the 2 parental strands to make them
available as template strands
 The untwisting process causes strain ahead of the
replication fork & topoisomerase helps relieve this
strain
 After the separation of the 2 parental strands, a single–
strand binding protein binds to the unpaired DNA
strands to stabilize them until they serve as templates for
the synthesis of new complementary strands
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 Proofreading & Repairing DNA
 During DNA replication, DNA polymerases
proofread each nucleotide against its template as soon
as it is added to the growing strand
 The incorrectly paired nucleotide is removed & then
synthesis resumes
 Mismatched nucleotides sometimes escape
proofreading or arise after DNA synthesis is completed
 So, these mismatched nucleotides are repaired by
enzymes during mismatch repair process
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 Chemicals in the environment or in the cell can
change nucleotides and affecting the genetic
information (harmfully) or

 DNA bases may undergo spontaneous chemical

changes under normal cellular conditions, but
fortunately,
fortunately changes in DNA are usually corrected
before they become self–perpetuating mutations

 Each cell continuously monitors & repairs its

genetic material
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 ~ 130 different DNA repair enzymes have been
identified so far in humans

 The segment of the strand containing the damage is

excised by a nuclease (DNA–cutting enzyme)

 Then, DNA polymerase and ligase fill in the gap

with nucleotides properly paired with the nucleotides
in the undamaged strand

 This kind of DNA repair is called nucleotide

excision repair
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 DNA repair enzymes in
human skin cells repair genetic
damage caused by UV rays of
sunlight, which is damaging the
thymine bases (thymine
(
dimers) that is causing DNA to
collapse and interfere with DNA
replication
 This damage gives disorder
xeroderma pigmentosum (in
most cases is caused by an
inherited defect in a nucleotide
excision repair enzyme)

Affected individuals are hypersensitive to sunlight and

may get skin cancer
 Replicating the Ends of DNA
Molecules
 In eukaryote, DNA polymerases cannot
replicate/repair a small portion of the DNA
 Because DNA polymerase only add nucleotides
to the 3′ end of a preexisting polynucleotide
 So, the replication machinery cannot complete
the 5′ ends of daughter DNA strands
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 Even if an Okazaki fragment can be started
with an RNA primer bound to the very end of
the template strand

 Because once that primer is removed, it cannot be

replaced with DNA

 Since there is no 3′ end onto which DNA

polymerase can add DNA nucleotides

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 So, repeated rounds of
replication produce shorter &
shorter DNA molecules

 Prokaryotes do not have this

problem because their DNA is
circular (with no ends)

Eukaryotic chromosomal
DNA molecules have nucleotide
sequences called telomeres at
their ends
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 Telomeres do not contain genes; the DNA consists of
multiple repetitions of one short nucleotide sequence

 In human telomeres,
telomeres the repeated unit is TTAGGG

 Telomere protects the genes from being eroded through

successive rounds of DNA replication
 In addition, telomere & specific proteins
associated with it prevent the staggered ends
of the daughter molecule from activating the
cell′s systems for monitoring DNA damage

 The end of a DNA molecule that is “seen” as a

double–strand break may otherwise trigger
signal transduction pathways leading to cell
cycle arrest or cell death
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 Telomeres do not prevent the shortening of DNA
molecules due to replication; they just postpone the
erosion of genes near the ends of DNA molecules
 Telomeres become shorter during replication
 This shortening can be connected to the aging
process!
 Telomerase catalyzes the lengthening of telomeres
to restore their original length & compensate for the
shortening that occurs during DNA replication

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 Normal shortening of telomeres
protects organisms from cancer by
limiting the number of cell divisions

 Further shortening would presumably

lead to self–destruction of the cancer

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How Eukaryotic Genomes Work
 In all organisms, DNA associates with proteins that
condense it

 In eukaryotes, the DNA–protein complex,

complex called
chromatin

 Chromatin undergoes condensation process

during the cell cycle to give the characteristic of the
chromosomes (short & thick) to distinguish them from
each other
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 Proteins called histones are
responsible for the first
level of DNA packing in
chromatin

 The mass of histone in chromatin is approximately

equal to the mass of DNA

 Histones have a high proportion of positively charged

amino acids (lysine & arginine), which bind tightly to the
negatively charged DNA
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 Unfolded chromatin has the appearance of beads on
a string
 Each “bead” is a nucleosome,
nucleosome the basic unit of DNA
packing
 The string between the beads is called linker DNA
 A nucleosome consists of DNA around a protein core
 The protein core composed of 2 molecules each of 4
types of histone: H2A, H2B, H3 & H4
 The amino end of each histone protein (histone tail)
extends outward from the nucleosome

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 A fifth histone, called H1, attaches to the DNA near
the nucleosome when a chromatin fiber undergoes the
next level of packing (condensation)
 The association of DNA & histones in nucleosomes
remains intact throughout the cell cycle
 The histones leave the DNA only transiently during
DNA replication & with very few exceptions, they stay
with the DNA during transcription
 The next level of packing is due to interactions
between the histone tails of one nucleosome & the linker
DNA & nucleosomes to either side
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 Types of chromatin: heterochromatin is
more compacted than euchromatin (“true
chromatin”)

 Because of its compaction, heterochromatin

DNA is largely inaccessible to transcription
enzymes & thus generally is not transcribed

 In contrast, the looser packing of

euchromatin makes its DNA accessible to
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