Can DNA Be Repaired?

A fundamental difference from RNA, protein,

lipid, etc.
• All these others can be replaced, but DNA
must be preserved
• Cells require a means for repair of missing,
altered or incorrect bases, bulges due to
insertion or deletion, UV-induced pyrimidine
dimers, strand breaks or cross-links
• Two principal mechanisms: mismatch repair
and methods for reversing chemical
damage
Chemistry 40

Mismatch Repair

• Mismatch repair systems scan DNA

duplexes for mismatched bases, excise
the mispaired region and replace it
• Methyl-directed pathway of E. coli is an
example
• Since methylation occurs post-replication,
repair proteins identify methylated strand
as parent, remove mismatched bases on
other strand and replace them

Chemistry 40

1
UVA irradiation causes dimerization of adjacent thymine bases. A cyclobutyl ring is formed
between carbons 5 and 6 of the pyrimidine rings. Normal base pairing is disrupted by the
presence of such dimers.

Chemistry 40

Reversing Chemical Damage

• Pyrimidine dimers can be repaired by

photolyase
• Excision repair: DNA glycosylase removes
damaged base, creating an "AP site"
• AP endonuclease cleaves backbone,
exonuclease removes several residues
and gap is repaired by DNA polymerase
and DNA ligase

Chemistry 40

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Base excision repair. A damaged base (■) is excised from the sugar-phosphate
backbone by DNA glycosylase, creating an AP site. Then, an apurinic/apyrimidinic
endonuclease severs the DNA strand, and an excision nuclease removes the AP site
and several nucleotides. DNA polymerase I and DNA ligase then repair the gap.

Chemistry 40

Base excision repair. A damaged base

(■) is excised from the sugar-phosphate
backbone by DNA glycosylase, creating
an AP site. Then, an
apurinic/apyrimidinic endonuclease
severs the DNA strand, and an excision
nuclease removes the AP site and
several nucleotides. DNA polymerase I
and DNA ligase then repair the gap.

Chemistry 40

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Base excision repair. A damaged base
(■) is excised from the sugar-phosphate
backbone by DNA glycosylase, creating
an AP site. Then, an
apurinic/apyrimidinic endonuclease
severs the DNA strand, and an excision
nuclease removes the AP site and
several nucleotides. DNA polymerase I
and DNA ligase then repair the gap.

Chemistry 40

Base excision repair. A damaged base

(■) is excised from the sugar-phosphate
backbone by DNA glycosylase, creating
an AP site. Then, an
apurinic/apyrimidinic endonuclease
severs the DNA strand, and an excision
nuclease removes the AP site and
several nucleotides. DNA polymerase I
and DNA ligase then repair the gap.

Chemistry 40

4
 What Is the Molecular Basis of Mutation?

• Point mutations arise by inappropriate base-

pairing
• Mutations can be caused by base analogs
• Chemical mutagens react with bases in DNA
• Insertions and deletions

Chemistry 40

MUTANT X

Chemistry 40

5
 transition
transversion

Chemistry 40

Point mutations due to base mispairings. (a) An example based on tautomeric properties. The
rare imino tautomer of adenine base pairs cytosine rather than thymine. (1) The normal A-T
base pair. (2) The A*-C base pair is possible for the adenine tautomer in which a proton has
been transferred from the 6-NH2 of adenine to N-1. (3) Pairing of C with the imino tautomer of A
(A*) leads to a transition mutation (A-T to G-C) appearing in the next generation. (b) A in the
syn conformation pairing with G (G is in the usual anti conformation). (c) T and C form a base
pair by H-bonding interactions medicated by a water molecule.

Chemistry 40

6
 Chemistry 40

DNA Techniques

Chemistry 40

7
 Agarose Gel Electrophoresis

technique used in the laboratory that

results in the separation of charged
molecules

Chemistry 40

In the case of DNA we can separate the molecules based

on their size. DNA has a negative charge in solution, so it
will migrate to the positive pole in an electric field.
Chemistry 40

8
 Southern Blotting

developed in l975 by
Edward M. Southern at
the University of
Edinburgh

makes use of
radiolabelled probes
that will hybridize with
a specific sequence of
DNA

Chemistry 40

DNA Markers
SNP- single nucleotide polymorphism
most frequent

RFLP- restriction fragment length polymorphism

RAPD – random amplified polymorphism

SSCP- single stranded chain conformation

polymorphism

STD- short tandem DNA

Chemistry 40

9
 DNA Markers

Mitochondrial DNA Analysis

older biological samples that lack nucleated cellular
material, such as hair, bones, and teeth, cannot be
analyzed with STR and RFLP

Y-Chromosome Analysis
especially useful for tracing relationships among males
or for analyzing biological evidence involving multiple
male contributors

Chemistry 40

Northern Blotting

detection of
specific RNA
sequences.

developed by
James Alwine and
George Stark at
Stanford University
and was named
such by analogy to
Southern blotting.

Chemistry 40

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 Western Blotting

allows
investigators to
determine, with a
specific primary
antibody, the
relative amounts of
the protein present
in different samples

Chemistry 40

What Does It Mean: “To Cloning”?

Clone: a collection of molecules or cells, all
identical to an original molecule or cell

• To "clone a gene" is to make many copies of

it - for example, in a population of bacteria
• Gene can be an exact copy of a natural gene
• Gene can be an altered version of a natural
gene
• Recombinant DNA technology makes it
possible
Chemistry 40

11
 Plasmids

Naturally occurring extrachromosomal DNA

• Plasmids are circular dsDNA

• Plasmids can be cleaved by restriction
enzymes, leaving sticky ends
• Artificial plasmids can be constructed by
linking new DNA fragments to the sticky
ends of plasmid
Chemistry 40

One of the first widely

used cloning vectors,
the plasmid pBR322.
This 4363-bp plasmid
contains an origin of
replication (ori) and
genes encoding
resistance to the drugs
ampicillin (ampr)and
Tetracycline (tetr). The
locations of restriction
endonuclease
cleavage sites are
indicated.

Chemistry 40

12
 Chimeric Plasmids
Named for mythological beasts with body
parts from several creatures
• After cleavage of a plasmid with a restriction
enzyme, a foreign DNA fragment can be
inserted
• Ends of the plasmid/fragment are closed to
form a "recombinant plasmid"
• Plasmid can replicate when placed in a
suitable bacterial host

Chemistry 40

Restriction endonuclease EcoRI cleaves double-

stranded DNA. The recognition site for EcoRI is the
hexameric sequence GAATTC:
5' . . . NpNpNpNpGpApApTpTpCpNpNpNpNp . . . 3'
3' . . . NpNpNpNpCpTpTpApApGpNpNpNpNp . . . 5'
Cleavage occurs at the G residue on each strand so that
the DNA is cut in a staggered fashion, leaving 5'-
overhanging single-stranded ends (sticky ends):
5' . . . NpNpNpNpG pApApTpTpCpNpNpNpNp . . . 3'
3' . . . NpNpNpNpCpTpTpApAp GpNpNpNpNp . . . 5'
An EcoRI restriction fragment of foreign DNA can be
inserted into a plasmid having an EcoRI cloning site by
(a) cutting the plasmid at this site with EcoRI , annealing
the linearized plasmid with the EcoRI foreign DNA
fragment, and (b) sealing the nicks with DNA ligase .

Chemistry 40

13
What Is the Polymerase Chain Reaction
(PCR)?

What if you don't have enough DNA for colony

hybridization or Southern blots?

• The small sample of DNA serves as template for

DNA polymerase
• Make complementary primers
• Add primers in more than 1000-fold excess
• Heat to make ssDNA, then cool
• Run DNA polymerase (usually Taq)
• Repeat heating, cooling, polymerase cycle

Chemistry 40

Chemistry 40

14
Chemistry 40

Chemistry 40
Fig. 10-1, p.241

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