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DNA Damage
Figure 5-46. A summary of spontaneous alterations likely to require DNA
repair. The sites on each nucleotide that are known to be modified by
spontaneous oxidative damage (red arrows), hydrolytic attack (blue arrows),
and uncontrolled methylation by the methyl group donor S-adenosylmethionine
(green arrows) are shown, with the width of each arrow indicating the relative
frequency of each event. (After T. Lindahl, Nature 362:709–715, 1993. ©
Macmillan Magazines Ltd.)
Deamination of Cytosine
Thyamine dimers
Nucleases
• Cleave nucleotide sequences
• DNases and RNases and non specific
nucleases
• ss and ds specificity
• Exonucleases (remove nucleotide from the
end)
• Endonucleases (recognize palindromic ds
DNA sequences)
Restriction endonucleases
• Three types (I, II, and III) – I and III require
ATP
• Type II are used as common molecular
biology tools
Type II restriction enzymes
• Recognize and cleave particular sequences
For example, BamHI
GGATCC
5’-N-N-N-N-G-G-A-T-C-C-N-N-N-N-3’
3’-N-N-N-N-C-C-T-A-G-G-N-N-N-N-5’
BamHI
5’-N-N-N-N-G-G-A-T-C-C-N-N-N-N-3’
3’-N-N-N-N-C-C-T-A-G-G-N-N-N-N-5’
5’-N-N-N-N-G
G-A-T-C-C-N-N-N-N-3’
3’-N-N-N-N-C-C-T-A-G
G-N-N-N-N-5’
“sticky ends” – overhanging sequence
Why do bacteria have
endonucleases?
How do they avoid digesting their
own DNA?
Overview
• DNA structure
– A, B, and Z DNA
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DNA intercelators and groove binders
Thermal melting of DNA
DNA tertiary structure
DNA methylation
DNA damage
nucleases
Which of the following statements correctly
describes B-DNA
A. B-DNA is usually found in solutions of
reduced water
B. B-DNA displays a wider helix in
comparison to Z-DNA
C. B-DNA forms a grooved left-handed helix
D. B-DNA has a helix shorter and wider
than A-DNA
RNA
Arrangement in three dimensions
Beyond the Four Bases
Elements of RNA Secondary Structure
Elements of RNA Tertiary Structure
Messenger RNA (mRNA)
•
•
Transcription product of DNA
In prokaryotes, a single mRNA contains the information for synthesis of many
proteins
In eukaryotes, a single mRNA codes for just one protein, but structure is composed of
introns and exons
Eukaryotic mRNA 5’ Cap
A phosphate is released by hydrolysis. The diphosphate
5′ end then attacks the α-phosphorus atom of GTP to
form a very unusual 5′-5′ triphosphate linkage. This
distinctive terminus is called a cap .
The N-7 nitrogen of the terminal guanine is then
methylated by S-adenosylmethionine to form cap 0. The
adjacent riboses may be methylated to form cap 1 or cap
2.
Caps contribute to the stability of mRNAs by
protecting their 5′ ends from phosphatases and
nucleases. In addition, caps enhance the translation
of mRNA by eukaryotic proteinsynthesizing systems
Eukaryotic mRNA poly A tail
mRNA molecule devoid of a poly(A) tail is usually a much less
effective template for protein synthesis than is one with a poly(A)
tail. – enhances translation of mRNA
The half-life of an mRNA molecule may also be determined in part
by the rate of degradation of its poly(A) tail. – enhances stability of
mRNA
Transfer RNA (tRNA)
• Recruits amino acid to the ribosome to
synthesize protein
• Extensive H-bonding creates four
double helical domains, three capped
by loops, one by a stem
• Many non-canonical base pairs found in
tRNA
Secondary Structure of tRNA
R = amino acid
Tertiary Structure of tRNA
5’
3’
Noncanonical base pairing and unusual
bases in tRNA
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Ribosomal RNA
Facilitate protein synthesis
Ribosomes are about 2/3 RNA, 1/3 protein
rRNA serves as a scaffold for ribosomal proteins
23S rRNA in E. coli is the peptidyl transferase –
catalytic! RIBOZYME
Secondary structure of rRNA
Small nuclear RNA (snRNA)
Participate in splicing the hnRNA to form the mature mRNA
snRNP
Size of
Role
snRNA(nucleotides)
U1
165
Binds the 5′ splice site and then the 3′ splice site
U2
185
Binds the branch site and forms part of the
catalytic center
U5
116
Binds the 5′ splice site
U4
145
Masks the catalytic activity of U6
U6
106
Catalyzes splicing
These RNA molecules and proteins assemble to form the Splicesome
snRNA catalysis
Hammerhead – catalytic RNA
Self cleaving RNA involved in replication of single stranded viroid (RNA
infectious agents of plant disease)
Small interfering RNA (siRNA)
•
Variety of roles in biology – most characterized is the interference of the
expression (translation) of a specific gene
RISC - RNA-induced silencing complexes
DNA & RNA Differences?
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Why is DNA 2'-deoxy and RNA is not?
Vicinal -OH groups (2' and 3') in RNA
make it more susceptible to hydrolysis
DNA, lacking 2'-OH is more stable
This makes sense - the genetic material
must be more stable
RNA is designed to be used and then
broken down
Hydrolysis of Nucleic Acids
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RNA is resistant to dilute acid
DNA is depurinated by dilute acid
DNA is not susceptible to base
RNA is hydrolyzed by dilute base
RNA World
Chemical view:
Abiotic nucleotide chemistry → RNA-catalyzed biochemistry.
Biochemical view:
RNA-based life → Protein/DNA-based life.
Why RNA vs Peptide or DNA
How could protein synthesis work
without protein and DNA?
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Polypeptides would have played only a limited role early in the evolution of life because their structures are not
suited to self-replication in the way that nucleic acid structures are. However, polypeptides could have been
included in evolutionary processes indirectly. For example, if the properties of a particular polypeptide favored the
survival and replication of a class of RNA molecules, then these RNA molecules could have evolved ribozyme
activities that promoted the synthesis of that polypeptide. This method of producing polypeptides with specific
amino acid sequences has several limitations. First, it seems likely that only relatively short specific polypeptides
could have been produced in this manner. Second, it would have been difficult to accurately link the particular
amino acids in the polypeptide in a reproducible manner. Finally, a different ribozyme would have been required
for each polypeptide. A critical point in evolution was reached when an apparatus for polypeptide synthesis
developed that allowed the sequence of bases in an RNA molecule to directly dictate the sequence of amino acids
in a polypeptide. A code evolved that established a relation between a specific sequence of three bases in RNA
and an amino acid. We now call this set of three-base combinations, each encoding an amino acid, the genetic
code. A decoding, or translation, system exists today as the ribosome and associated factors that are responsible
for essentially all polypeptide synthesis from RNA templates in modern organisms. The essence of this mode of
polypeptide synthesis is illustrated in Figure 2.8.
An RNA molecule (messenger RNA, or mRNA), containing in its base sequence the information that specifies a
particular protein, acts as a template to direct the synthesis of the polypeptide. Each amino acid is brought to the
template attached to an adapter molecule specific to that amino acid. These adapters are specialized RNA
molecules (called transfer RNAs or tRNAs). After initiation of the polypeptide chain, a tRNA molecule with its
associated amino acid binds to the template through specific Watson-Crick base-pairing interactions. Two such
molecules bind to the ribosome and peptide-bond formation is catalyzed by an RNA component (called ribosomal
RNA or rRNA) of the ribosome. The first RNA departs (with neither the polypeptide chain nor an amino acid
attached) and another tRNA with its associated amino acid bonds to the ribosome. The growing polypeptide chain
is transferred to this newly bound amino acid with the formation of a new peptide bond. This cycle then repeats
itself. This scheme allows the sequence of the RNA template to encode the sequence of the polypeptide and
thereby makes possible the production of long polypeptides with specified sequences. The mechanism of protein
synthesis will be discussed in Chapter 29. Importantly, the ribosome is composed largely of RNA and is a highly
sophisticated ribozyme, suggesting that it might be a surviving relic of the RNA world.