Nucleic Acid Structure &
Replication
Dr. Bart Dzudzor
Overview of organizations of life in Eukaryotes
• Nucleus = library
• Chromosomes = bookshelves
• Genes = books
• Almost every cell in an organism contains the same libraries and the
same sets of books.
• Books represent all the information (DNA) that every cell in the body
needs so it can grow and carry out its various functions.
Nucleotide Structure
Base
Phosphates
Purine or Pyrimidine
1, 2, or 3
Sugar
Ribose or Deoxyribose
Nucleoside
Nucleotide
A nucleotide: Pentose+phosphate+base
OH
CH2O OH
2’
3’
OH OH
Ribose
RNA
OH
CH2O OH
3’
2’
OH H
Deoxyribose
(2’deoxy)
DNA
- The presence of the 2’OH confers special chemical and
structural properties to RNA compared to DNA
Major differences between A and B-DNA
1) A-DNA is shorter due to
different sugar pucker
2) Bases shifted away from
helical axis in A-DNA:
a) Results in cavernous
major groove and
shallow minor groove
b) Results in 6 Å hole
3) Base pairs dramatically
tilted in A-DNA
Relative stability of A- vs. B-form helices
DNA
1. In aqueous solution, B-DNA is
favored over A-DNA, apparently due
to B-DNA’s “spine of hydration”.
2. Reduced water activity in solutions
containing high concentrations of
organic solvents (or in partially dried
out DNA fibers favors A-DNA.
RNA - Steric crowding with 2’-OH group in
the C2’ endo sugar pucker forces RNA
double helices into A-conformation
Z- DNA
• Occurs in DNA sequences with stretches of consecutive G-C base pairs
• Left- handed helix
• Jagged backbone
Zig zag backbone
Hairpin loops in transcription
termination process can promote
the formation of Z - DNA
• Requires high salt
• G nucleotides switch from C2’ endo to C3’ endo and no change in C
nucleotide sugar pucker.
Major types of RNA in Humans
• mRNA
• rRNA
• tRNA
• The primary structure of RNA is defined as the number and sequence
of ribonucleotides in the chain.
A mature Eukaryotic mRNA
After Processing
m7GpppN1
CAP
Structure
AUG
stop
AAAAAAAA
Poly(A) tail
Red elements (Cap, polyA tail) are not encoded within
The genes: they are added after transcription
tRNAs are adaptor molecules
Crick’s
Adaptor
Hypothesis
DNA Replication and Repair
Dr. Bart Dzudzor
UGMS
DNA replication is Semiconservative
The two old DNA strands serves as a template
for the formation of an entire new strand.
DNA polymerization
5’primer3’
3’
template
5’
3’
New
strand
5’
+ dNTPs
3’
5’
+ PPi
3 properties common to ALL
DNA polymerases
1) Catalyze the
polymerization of
deoxyribonucleotides
in the 5’ to 3’
direction.
2) Require a template.
3) Require a primer
Biological roles of Pol I:
•
•
•
•
removes RNA primers
fills gap with DNA
DNA repair
5’→3’ polymerase fills gap left by repair enzymes which
excise regions of DNA containing damaged or mispaired
nucleotides (more later)
• Processivity: usually catalyzes ~20 nt additions before falling
off template.
Enymatic activities of DNA Pol I
• 5’ -> 3’ Polymerase
• 3’ -> 5’ Exonuclease
• 5’ -> 3’ Exonuclease
Fidelity of DNA replication
NonWatson/Crick
geometry
(Wobble pair)
Watson/Crick
geometry, but
rare tautomer
Low error frequency (≈ 10-9) accounted for by redundant
safeguards.
1. Binding pocket of DNA polymerase clamps tightly around
the base before catalysis occurs. Wobble pairs don’t fit
and so catalysis can’t occur.
• But binding pocket is unlikely to be rigid enough to
exclude wobble pairs every time.
2. Enol tautomers are very unstable (keto/enol
tautomerization equilbrium constants are in the 10-5 to
10-3 range).
• But this isn’t enough to account for the 10-9 error
frequency.
3. DNA polymerases have “editing exonuclease activities”
that allow them to erase mistakes and try again
4. Cells contain mismatch repair systems that come along
after DNA polymerase to clean up any residual errors.
Editing by the 3’->5’ exonuclease activity
5’
3’
5’
template
Polymerization
5’
5’
3’
3’->5’ exo triggered
by the mistake
5’
5’
3’
3’->5’ exo
5’
3’
template
New
stran
d
5’
Polymerization
3’
5’
5’
Editing of mistakes require a switch between :
polymerization mode and editing mode
Two problems posed by the properties
of the known DNA polymerases
1.
The directionality problem. How can
DNA polymerase replicate both strands
behind each replication fork, when all
polymerases operate in the 5’ to 3’
direction?

2.
Solution - semidiscontinuous DNA synthesis
The priming problem. Since all DNA
polymerases require a primer (usually of
at least 10 nucleotides in length), where
do the primers come from?

Solution - primers are made of RNA
The bidirectionality problem
Synthesis of DNA on the
Synthesis of DNA
lagging strand requires
is semi-discontinuous
continuous synthesis
of primers
In bacteria, the Okazaki fragments are about 1000-2000 bp in length
The replisome of E.coli prokaryotes
1) Helicases
Unwind DNA at the replication
fork in a reaction coupled
to ATP Hydrolysis
2) Single-stranded DNA
binding proteins (SSB)
Bind and stabilize the DNA in a
single stranded conformation
after the melting by helicases
3) The Primosome
Synthesizes RNA primers
of the lagging strand
Contains Primase
4) DNA Polymerase III :
The replicase
5) DNA topoisomerase II
Relaxes supercoiled DNAalso has ligase
that forms ahead of the activity
replication fork. Decatenates
the final product
6) DNA Polymerase I
Replaces RNA primers with
DNA by nick translation
7) DNA Ligase
Joins the Okazaki
fragments
Unwinding the duplex at the replication
fork
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
T7 Gene 4 Helicase
Sliding b clamps ensure processivity of DNA
polymerase III
Polymerase III
3’
b- Clamps
Newly
Replicated
Strand
B clamp pushes the DNA Pol III
This increases procesivity
5’
Template Strand
3’
How do we get clamp on and off?
The replisome of E.coli in action: Coordination
of Leading and Lagging Strand synthesis
Summary of DNA replication paradigms for prokaryotes
• Semiconservative
• Bidirectional
• One origin per bacterial chromosome
• Semidiscontinuous
• RNA primed
The Eukaryotic Replisome
• Pol  - the eukaryotic replicase
The eukaryotic
replisome is
homologous in
many respects to the
bacterial
replisome!!!
• Pol /primase - contains both
primase and DNA polymerase
activities
• PCNA - trimeric sliding clamp
• Replication Factor C (RFC) - the
clamp loader
• MCMs - a heterohexameric
helicase
• Replication Protein A (RPA) =
SSB
• RNase H - nuclease that is
specific for RNA in RNA/DNA
hybrids - excises primers
Replication of the ends of linear
chromosomes
One end of a
chromosome
Primase
Pol 
+
3’
5’
3’ RNase H
Pol 
Ligase
5’
Without a way to fill in
this gap, information will
be lost from the ends of
the chromosomes at each
cell cycle!!
This will eventually lead
to cell death!!!
telomerase
Primase
Pol 
Ligase
RNase H
Telomerase--aging, cancer, and disease
Most somatic cells have low or undetectable level of
telomerase activity.
Telomere length is correlated with cellular aging.
Telomerase is active in some germline, epithelial, and
stem cells (haemopoeitic cells), and in >90% of cancer cell
lines.
Mutations in the RNA component of human telomerase
have been linked to autosomal dominant dyskeratosis
congenita and some forms of aplastic anemia.
Telomerase is required for telomere maintenance.
Telomerase is responsible for the immortal phenotype of
cancer cells.
After a few generations,
telomerase-mutant mice exhibit
reduced fertility, signs of
premature aging, and
shortened
life-span.
Psychological stress leads to reduced telomerase
activity and increased telomere shortening! This
presumably results in premature aging!!
Telomere length
Telomerase activity
Study conducted on mothers
under stress due to need to
care for a chronically ill
child.
• Telomere shortening in high
stress subjects is the
equivalent of one decade of
additional aging!
Summary of DNA Replication
•
•
•
•
•
•
•
•
Identification of the initiation site of replication (OriC)
Unwinding of parental DNA (DS -> SSDNA)
Formation of replication fork
Synthesis of RNA primer complementary to the DNA template by primase
Leading strand is synthesized in the 5’-3’ direction by DNA Pol.
Lagging strand is synthesized as Okazaki fragments
RNA primers removed when polymerization is done
The gaps are filled by dNTPs and the pieces are joined by DNA ligase which
requires energy from ATP.
DNA damage
A gallery of horrors
• 1. UV damage
• 2. Environmental Chemicals
e.g. alkylating agents
• 3. Normal Physiological agents
H20 (Hydrolytic deamination)
depurination
02 Oxidation)
nitrites (Oxidative deamination)
Alkylation
- 4. Replication errors (wrong base)
• Et cetera
UV light: Ultraviolet radiation
causes damage in DNA by
cross-linking of the adjacent
pyrimidine bases to form dimers.
For example, the cross-linking of
adjacent thymine to form thymine
dimers results in the inability of
DNA to replicate properly
c-c =cytosine dimer leading to
DNA Damage
Repair is by
1 Direct Reversal of Damage
• Photolyase reversion of
Y(Pyrimidine ) dimers
various DNA Damages that need to be repaired:
-Pyrimidine dimers
- Alkylation of bases
(UV light)
Methylation of guanine N6
G-> O6meG
- Hydrolysis of glycosidic bond
(Depurination)
Direct repair is done here by
Dealkylation of guanines by suicidal
MGMTase
Dealkylation of 1mA and 3mC by AlkB
- Deamination of bases - Oxidative damages
•Spontaneous
•G -> 8 oxoguanine
•Chemically induced
•Strand Break
Oxdative damage results in DNA Damage when
•C->U
reactive oxygen species(ROS) which are chemical
agents convert guanine to 8 oxoguanine
•5 meC -> T
•A->HX
Repair here is done by
2 Nucleotide excision repair
Where in Bacteria it uses the uvr
system : UvrA, UvrB, UvrC,
Helicase II (UvrD) They also use
DNA pol. I, DNA ligase
In Eukaryotes they use
Xeroderma pigmentosum
proteins and TFIIH
Base excision repair by two enzymes
• Uracil-N glycosylase •
8-oxoG glycosylase
oxidative damage results in DNA Damage when reactive oxygen species(ROS)
which are chemical agents convert guanine to 8 oxoguanine
Repair here is done by
Base excision repair by two enzymes
• Uracil-N glycosylase •
8-oxoG glycosylase
Uracil N-glycosylase (UNG) and 8-oxoguanine DNA glycosylase (OGG1) are both
DNA repair enzymes that play important roles in protecting cells from damage
caused by reactive oxygen species (ROS).
UNG removes uracil residues from DNA.
Uracil is not normally found in DNA, but it can be produced by the deamination of
cytosine.
If uracil is not removed from DNA, it can mispair with adenine during DNA
replication, leading to mutations.
OGG1 removes 8-oxoguanine (8-oxoG) residues from DNA.
8-oxoG is a guanine base that is damaged by ROS.
If 8-oxoG is not removed from DNA, it can mispair with cytosine during DNA
replication, leading to mutations.
Both UNG and OGG1 are members of the glycosylase family of enzymes.
Glycosylases catalyze the cleavage of the glycosidic bond between the base and
the sugar of a nucleotide.
UNG and OGG1 are essential for maintaining genomic stability and preventing
cancer.
Mutations in the genes that encode UNG and OGG1 have been linked to an
increased risk of cancer
Induction of Pyrimidine Dimers
by UV light
Hydrolytic or oxidative deamination
Nitrosonium ion
•Electron hungry
•Formed from: nitrates and nitrites (common food preservatives,
but also naturally occurring in foods such as spinach)
•Also formed from nitrosamines (byproducts of rubber
production)
So processed
foods are
dangerous.
But so is water!
Spontaneous Deaminations
C --> U
10-7/24 hours:
100 events/day
for a mammalian
cell
A --> H
10-9/24 hours
G --> X
10-9/24 hours
•
Oxidative damage of DNA
- Source of oxidative agents
Consequences for Nucleotides:
Respiratory Chain:
O2
O
CytC oxidase
2H2O
+H O1e--OH + .OH
2H
2 2
O2
1e-
• Fenton Chemistry (Metals)
N
N
N
H
N
Guanine H2
N
- Neutralization of reactive species Thymine
2O2+2H+
2H2
O2
.
OH
superoxide
H2O2
dismutase
Deoxyribose
+O2
catalase
2H2O+ O2
No cellular
Neutralization !!!
-> Main source of
Oxidative agent
H
N
H2O2
O
-OH
O
N
H
N
N N
8-oxo H2
Guanine
5-formyl
Uracil
Ribose
Consequences for Nucleic Acids:
Strand Breaks
(bad for DNA replication)
Consequences of O6-meG For Replication :
How to minimize damage to your DNA
• Avoid chemical warfare
• Avoid processed foods
• Avoid the highways
• Avoid sunlight
• Avoid aerobic activities
• Avoid water
But if all these precautions fail, our cells
have multiple DNA repair pathways to undo
the damage!!!
1- Direct Reversal of Damage
• Photolyase reversion of Y dimers
• Dealkylation of guanines by suicidal MGMTase
• Dealkylation of 1mA and 3mC by AlkB
2- Base excision repair
• Uracil-N glycosylase
• 8-oxoG glycosylase
3- Nucleotide excision repair
DNA repair
Strategies
& Enzymes
• Bacteria: UvrA, UvrB, UvrC, Helicase II (UvrD)
• DNA pol. I, DNA ligase
• Eukaryotes : Xeroderma pigmentosum proteins,
TFIIH
4- Methyl directed Mismatch repair
• MutS, MutL, MutH
Mismatch repair
Eukaryotes
• Similar mismatch repair system contains MutL and MutS
homologues.
• In humans, mutations in these genes are responsible for
Hereditary nonpolyposis colorectal cancer ( HNPCC), an inherited
predisposition to colon cancer.
• Eukaryotes lack both Dam methylase and mutH
• It’s not known how the mismatch repair machinery differentiates
between the parental and daughter strands.