We Need Nucleic Acids!
Chapter 25
Homework Assignment
• The following problems will be due once we finish
the chapter:
4 5,
4,
5 8,
8 9,
9 11
DNA
Protein Protein
Trait
DNA RNA
mRNA
RNA
rRNA
Pol
tRNA
• DNA contains genes, the information needed to synthesize functional proteins
and RNAs
– DNA also
l contains
t i segments
t th
thatt play
l a role
l iin regulation
l ti off gene
expression (promoters, operators, etc.)
• Messenger RNAs (mRNAs) are transcribed from DNA by RNA polymerases
and carry genetic information from a gene to the ribosome complex
Minimal Coverage of Section 25.3
• Ribosomal RNAs (rRNAs) are components of the ribosomes and play a role
in protein synthesis in conjunction with the template mRNA and the AA
carrying tRNA
• Transfer RNAs (tRNAs) carry the AAs designated by the codons of the
mRNA and bind to the Ribosome to help form the growing polypepide chain
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Chapter 25
Nucleotides and Nucleic Acids
DNA Metabolism
Nucleotides are the building blocks of nucleic acids
A nitrogenous base
A phosphate group
(pyrimidines or purine)
A pentose sugar
DNA Polymerase III
Nucleotides have three characteristic components
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1
Structure of a Nucleotide
Nucleic Acid Structure
Major Bases in Nucleic Acids
DNA Strands
• The bases are abbreviated by
their first letters (A, G, C, T, U).
• The purines (A, G) occur in
both RNA and DNA
• The opposing strands of DNA are not identical, but
are complementary.
• This means: they are positioned to align
complementary base pairs:
– C with G, and A with T, but the strands run antiparallel to
each other
• You can thus predict the sequence of one strand
given the sequence of its complementary strand.
• The pyrimidine C occurs in
both RNA and DNA, but
• Note that sequence conventionally is written from
the 5' to 3' end
• T occurs only in DNA, and U
occurs only in RNA
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• Such a structure is useful for information storage
and transfer!
5
Chapter 25
Nucleic Acid Structure
Nucleic Acid Structure
Stabilization of Double Helix
DNA
• Weak forces stabilize the double helix:
• DNA consists of two helical
chains wound around the same
axis in a right-handed fashion
aligned in an antiparallel
fashion.
(1) Hydrophobic Effects: Burying purine and pyrimidine
rings in the interior of the helix excludes them from water
(2) Stacking interactions: Stacked base pairs form van der
W l contacts
Waals
t t
• There are 10.5 base pairs, or 36
Å, per turn of the helix.
(3) Hydrogen Bonds: H-bonding between the base pairs
(not on the backbone!)
• Alternating deoxyribose and
phosphate groups on the
backbone form the outside of the
helix.
(4) Charge-charge interactions: Electrostated repulsion of
negatively charged phosphate groups is decreased by
cations (e.g. Mg2+) and cationic proteins
• The planar purine and
pyrimidine bases of both strands
are stacked inside the helix.
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6
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2
DNA Metabolism
Nucleic Acid Structure
The BIG Picture
Interstrand H-bonding Between DNA Bases
• DNA is the molecular vehicle used for the stable
storage of genetic information
• Stable does not mean static however!
• DNA metabolism encompasses:
– Processes that yield faithful copies of DNA molecules
(aka. Replication)
– Processes that affect the inherent structure of the
information (aka. Repair and Recombination)
What do you think is the most important
requirement for any copy of DNA?
Watson-Crick base pairing
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DNA Metabolism
Nucleic Acid Structure
Gene Naming – An Aside
DNA Structure Summary
• Most of what we now know about the replication process
was gleaned by observation of bacterial systems (WHY?)
• In this chapter, you will see the names of numerous genes
and their products
• Bacterial genes are usually names using three lower case,
italicized letters that often reflect the gene
gene’s
s apparent
function.
• If more than one gene affects the same process the letters
A, B, C, etc. are added
– dnaA: Gene product that affects DNA replication
– recB: Gene product that affects recombination
• Bacterial proteins often retain the name of their genes,
except you capitalize the first letter and lose the italics
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DNA Metabolism
DNA Replication
Replication is Semi-conservative
• Similar to what we see in PCR, each strand of the
duplex DNA serves as a template for the synthesis
of a new DNA strand
Chapter 25
• The use of these “old” DNA strands as templates
produces two “new”
new DNA strands that are duplexed
with their template.
Chapter 26
• This process yields four DNA strands in two
“old/new” duplexes
Chapter 27
• This is semi-conservative replication
What do you think conservative replication would be?
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DNA Replication
DNA Replication
Replication Follows a Set of Fundamental Rules
Replication is Semi-conservative
• DNA replication follows a set of fundamental rules
that were determined based on early research with
bacterial DNA processes:
(1) Replication is semi-conservative
(2) Replication begins at an origin and usually
proceeds bidirectionally
• This hypothesis
(proposed by our good
friends Watson and
Crick) was verified by
Meselson and Stahl in
their CsCl gradient
experiment.
(3) DNA synthesis proceeds in a 5’ → 3’ direction
and is semi-discontinuous
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4
DNA Replication
DNA Replication
So how does Replication start and/or run?
Now the strands are open, what next?
• Once semi-conservatism was confirmed
several other questions came up:
– Are the parent DNA strands completely unwound
before replication?
– Does replication begin at random points or a
unique site?
– Once initiated, does replication move in uni- or
bidirectional directions?
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DNA Replication
DNA Replication
Replication begins at an Origin
DNA Synthesis Proceeds 5’ → 3’
• To be used as a template, the double helix
must first be opened up and the two strands
separated to expose unpaired bases.
• The positions at which the DNA helix is first
opened are called replication origins
• IIn simple
i l cells
ll lik
like th
those off b
bacteria
t i or yeast,
t
origins are specified by DNA sequences
several hundred nucleotide pairs in length.
• This DNA contains short sequences that
attract initiator proteins, as well as stretches of
DNA that are especially easy to open.
What bases do you think are present at the origin?
Chapter 25
• The opening of the duplex puts
a “bubble” of single stranded
DNA with two replication
forks ready for strand
y
synthesis
• John Carins demonstrated
experimentally that synthesis
of the new DNA strands is
simultaneous and
bidirectional
18
• A new strand of DNA is
always synthesized 5’ to 3’
with the new dNTP being
added to the 3’ – OH group
• The leading strand is the
template that is read from its
3’ end to its 5’ end.
• The lagging strand is read 5’ to 3’. What does that mean
for the synthesized strand? Uh oh.
• Reiki Okazaki determined that the lagging strand is in fact
synthesized discontinuously in pieces called Okazaki
fragments.
• These fragments are connected later by another enzyme.
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5
DNA Replication
DNA Replication
Who does all the work?
DNA Polymerase
• Enzymes of course!
• Nucleases:
• The general DNA Polymerase reaction is a nucleophilic
attack of the 3’ – OH group (the nucleophile) on the α
phosphate group of an incoming deoxynucleoside
– Catalyze the hydrolysis of DNA (DNases) or RNA (RNases)
Hydrolysis of PPi
will give us another
19 kJ/mol of energy
to push
p sh the reaction
– Either externally (exonucleases) or at internal sites (endonucleases)
• Polymerases:
– C
Catalyze
t l
th
the fformation
ti off a phosphodiester
h
h di t bond
b db
between
t
th
the 5’ –
phosphate and the 3’- OH
(dNMP)n + dNTP → (dNMP)n+1 + PPi
– Requires a primer, a short strand of DNA (or RNA) that has a free
3’ – OH group for elongation
• All the other players:
– Helicases, Ligases, Topoisomerases, DNA binding proteins,
and many more!
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DNA Replication
DNA Replication
DNA Polymerase
DNA Polymerase
• Prokaryotes and Eukaryotes actually possess several
distinct DNA Polymerases
• They all catalyze the same fundamental reaction, a
phosphoryl group transfer.
• There are two requirements for DNA polymerization:
– All DNA Polymerases
P l
require
i a template.
t
l t
– All DNA Polymerases require a primer with a free 3’ – OH group for
polymerization. Most primers are RNA that is synthesized by
specialized enzymes (e.g. DNA Primase)
• The average number of nucleotides added before a DNA
polymerase dissociates from a growing strand defines its
processivity
• DNA Polymerases vary greatly in this value.
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• Replication is VERY accurate.
Good thing!
• The pairing of bases depends
on more than the hydrogen
bonding patterns between the
bases.
• In addition, the active site of DNA polymerase
accommodates only base pairs with aligned
geometries.
• An incorrect dNTP may be able to hydrogen
bond with another base, but it generally will
not fit in the enzyme active site!
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6
DNA Replication
DNA Replication
DNA Polymerase
So, let’s replicate!
• Fit in the active site cannot account wholly for
the high fidelity of replication
• An incorrect base is still incorporated for
every 104 to 105 bases added.
• Almost all DNA polymerases have an
intrinsic, separate 3’ → 5’ exonuclease
activity that double-checks each dNTP after it
is added.
• This activity, known as proofreading, is not
simply the reverse of the polymerization
reaction.
• Taken with the base selection, DNA
Polymerase leaves behind one net error for
every 106 to 108 bases added.
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What needs to be done?
(1) Locate the Origin of replication
(2) Unwind the DNA (and continue to unwind it
as we move along the template!)
(3) We should also probably stabilize the
ssDNA structure
(4) Prime the DNA for the polymerase
(remember PCR?)
(5) Replicate the DNA
(6) Replace the primer (if it was RNA!)
(7) Fix the nicks
(8) Find the terminus sequence and stop
(9) CHECK OUR WORK!
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Initiation
Elongation
Termination
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DNA Replication
DNA Replication
DNA Polymerase
DNA Replicase System
• Replication in E. coli requires 20 or more different
enzymes and proteins, each performing a specific task.
• Taken together, the entire complex is called the DNA
Replicase System or the DNA Replisome
• Components include:
DNA Polymerase III
– Helicases – moves along the DNA template and separates
the strands using chemical energy (ATP)
– Topoisomerases – relieves topological stress induced by
strand separation.
– DNA-Binding proteins – stabilize the separated DNA strands
– Primases – synthesize the RNA primers on the template
– DNA ligases – seal nicks in the phosphodiester backbone
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DNA Replication
DNA Replication
Initiation
Elongation
• The replisome promotes rapid DNA
synthesis, adding ~ 1000 nucleotides
per second to each strand.
• Once the Okazaki fragment has be
completed, its RNA primer is removed
and replaced with DNA by DNA Pol I
• The remaining nick is sealed by DNA
Ligase
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Chapter
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DNA Replication
DNA Replication
Elongation
DNA Ligase
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DNA Replication
DNA Repair
Termination
Mutations
• Eventally, replication forks will meet at a terminus region containing
multiple copies of a 20 bp sequence called Ter
• The Ter sequence is a binding site for the Tus protein (terminus
utilization substance) and a Tus-Ter complex is formed.
• When a replication fork runs into a Tus-Ter complex, it stops.
• Replication halts when the second fork runs into the stopped fork
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• Unrepaired DNA damage (a lesion) can result in a change in the
base sequence of the DNA.
• If replicated, this change can be passed onto daughter cells and
become permanent, a mutation.
• The mutation can involve base substitutions or the addition/
deletion of one or more base pairs.
• If the mutation takes place in nonessential DNA or has a
negligible effect on gene function, it is termed a silent mutation.
• A typical mammalian genome accumulates 1000s of lesions in
24 hours
• Our repair systems manage to keep mutations at around 1 in
1000 lesions.
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DNA Replication
DNA Repair
Termination
Repair Systems
• Eventually, replication forks will meet at a terminus region
containing multiple copies of a 20 bp sequence called Ter
• The Ter sequence is a binding site for the Tus protein (terminus
utilization substance) and a Tus-Ter complex is formed.
• When a replication fork runs into a Tus-Ter complex, it stops.
• Replication halts when the second fork runs into the stopped fork
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DNA Repair
DNA Repair
Mismatch Repair
Base-Excision Repair
• Mismatches are corrected to reflect the
information of the original (template) strand.
• The repair system must be able to
discriminate between the new strand and the
template.
• In E. coli, the cell uses methylation of the
template strand to “tag” it as the original
strand.
• This “tagging” is catalyzed by Dam methylase
• Dam methylase methylates DNA at the N6
position of adenines within (5’)GATC
sequences.
• The “tagging” mechanism of other bacteria
and eukaryotes is still unknown!
Chapter 25
• The DNA Glycosylases recognize common
DNA lesions, such as products of cytosine and
adenine deamination, and remove the effected
base by cleaving the N-glycosyl bond (CHP 8)
• This cleavage leaves an apurinic or
apyrimidinic site in the DNA (aka. The AP site
or abasic
b i site)
it )
• Now, we need to repair the DNA. This is not
merely attaching an undamaged base:
– AP endonuclease cuts the backbone near the AP
site, marking the lesion.
– DNA Polymerse I removes a segment of DNA at
the AP site then synthesizes a new strand from the
free 3’ – OH.
– DNA Ligase then seals the remaining nick.
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DNA Repair
DNA Repair
Mismatch Repair
Nucleotide-Excision Repair
• DNA lesions can also cause large distortions of the helical structure of
the dsDNA (Cyclobutane pyrimidine dimers for example)
• These distortions are generally repaired by removing whole sections
of DNA and replacing it with newly synthesized DNA (aka. Nucleotide
- excision repair)
Exinuclease because it can
cleave at two sites within
the same strand
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DNA Repair
DNA Repair
Direct Repair
Recombinational DNA Repair
• Some damage can be repaired
in place.
• Once such process is catalyzed
by the DNA Photolyases
• These enzymes can utilize
energy derived from absorbed
light to reverse damage
produced from UV light
(Pyrimidine dimers!)
• The chromophore of these
enzymes is FADH- and a folate
• Placental animals (including
humans) do not have this class
of enzymes.
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•
A potentially dangerous type of DNA damage
occurs when both strands of the double helix
are broken, leaving no intact template strand
for repair.
•
If these lesions were left unrepaired, they
would quickly lead to the breakdown of
chromosomes into smaller fragments
•
The end-joining mechanism, which can be viewed as an emergency solution to the
repair of double-strand breaks, is a common outcome in mammalian cells. WHY
is this OK??!!
•
The second mechanism transfers nucleotide sequence information from the intact
DNA double helix of the homologous chromosome to the site of the double-strand
break in the broken helix.
•
A DNA replication process uses the undamaged chromosome as the template for
transferring genetic information to the broken chromosome, repairing it with no
change in the DNA sequence
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DNA Repair
Figure 5-53 Alberts et al. “Molecular Biology of the Cell” 4th Ed.
43
DNA Recombination
Direct Repair
• Enzymes are also available to repair
methylated bases.
• The AlkB protein is an α-KG-Fe2+dependent dioxygenase
• The rearrangement of genetic information within and among
DNA molecules can collectively be termed genetic
recombination
• These events can fall into three categories:
– Homologous Genetic Recombination – the exchange of genetic
information between any two DNA molecules (or segments) that share an
extended region of nearly identical sequence.
– Site-Specific Recombination – exchanges occur only at a particular DNA
sequence.
– DNA Transposition – usually involves a short segment of DNA with the
capacity to move from one location in a chromosome to another.
• The function of these systems include:
– DNA Repair; DNA Replication; Regulation of Gene Expression;
Chromosome segregation; Maintenance of genetic diversity; and
programmed genetic rearrangements during embryonic development.
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11
DNA Recombination
DNA Recombination
Homologous Genetic Recombination
•
Transposons
The homologous genetic recombination reaction is
essential for every proliferating cell because accidents
occur in nearly every round of replication that interrupt the
replication fork.
•
Recombination occurs with the highest frequency during
meiosis
•
Recombination occurs due to the need to keep the two
pairs of sister chromatids in close contact for even
distribution among the resulting haploid gametes
•
Recombination also allows for the movement of transposable elements
(aka. Transposons)
•
These segments of DNA are found in all cells and can “jump” from one
place on a chromosome to another on the same or a different
chromosome.
•
The new location is determined randomly, requiring tight regulation as
insertion of an element into a vital gene could result in cell death
death.
•
Movement is catalyzed by a Transposase that recognizes the terminal
repeats at the end of a transposon.
Once inserted into the new site, the
overhangs are paired by DNA
Polymerase and sealed by DNA
Ligase.
•
•
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This gives a short direct repeat of the
sequence on either side of the insert
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Figure 5-70 Alberts et al. “Molecular Biology of the Cell” 4th Ed.
47
Figure 5-55 Alberts et al. “Molecular Biology of the Cell” 4th Ed.
DNA Recombination
DNA Recombination
Site-Specific Recombination
Transposons - Immunoglobulins
•
Site-specific recombination is limited to
specific sequences and is catalyzed by a
system including enzymes and a unique
recognition site
•
A DNA Recombinase recognizes a
recombination site (20 to 200 bp sequence)
Chapter 25
• Transposition is an excellent way to
introduce diversity into a specific gene
area as long as it is regulated.
• One example of programmed
transposition is the generation of
complete immunoglobulin genes from
separate gene segments within the
genome.
• Like going to the iTunes (your genome)
and putting together your own playlist
(the final Ig) by selecting individual songs
(the transposable gene segments).
• Let’s us make millions of Igs without
maintaining millions of separate genes.
Cool!
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12
DNA Recombination
Transposons - Retroviruses
•
Outside the cell, a retrovirus
(like HIV) exists as a singlestranded RNA genome packed
into a protein capsid along with a
virus-encoded reverse
transcriptase enzyme.
•
Specific DNA sequences near
the two ends of the doublestranded DNA product produced
by reverse transcriptase are then
th
held together by a virus-encoded Figures 5-73 and 5-75 Alberts et al. “Molecular Biology of the Cell” 4 Ed.
integrase enzyme.
This integrase creates activated 3′-OH
viral DNA ends that can directly attack a
target DNA molecule through a
mechanism very similar to that used by the
cut-and-paste transposons.
•
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