Transcript Chemistry of Nucleic Acids

DNA Mutation
 Mutation is the process by which gene (chromosome)
changes structurally
 In 1943 Luria and Delbruck used the fluctuation test to
demonstrate that phenotypic variant in bacteria is due to
mutation
 Enrichment media in a perti dish was plated with E. coli in
presence of phage T1
 Under normal circumstances there will be no bacterial
colonies as all bacteria will be lysed
 When resistant bacterial grow
Genetic fine structure
Complementation
 If two recessive mutations arise independently and if they do
so there is there is complementation between the mutants
 To test complementation test mutant homozygotes are crossed
 If mutants are allelic (affect same gene) both copies of the
gene are mutant, resulting in a mutant phenotype
 If nonallelic there is a wide type phenotype

 Mutation that fails to complement each other are called
functional alleles
Figure 1. Complementation test to define allelism. (a) Shows
allelic mutants affecting same gene and (b) mutants are
nonallelic
Mutation
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Point mutations
 These consist of single changes in the nucleotide sequence and
they could be replacement, addition or deletion
 A second point mutation in the same gene can result in double
mutation, reversal to original or intragenic suppression
(addition of a base followed by deletion of a nearby base)
 Mutations will result in new amino acids and new proteins
appear that can alter the morphology or physiology of the
organism and result in phenotypically novel or lethal mutants
Figure 2. Types of point mutations on DNA showing singlestep (replacement, addition deletion) and double step (double
mutation, reversal to original or intragenic suppression
Frameshift mutations
 Point mutations that add or subtract a base are potentially the
most devastating because they change the reading from the site
of mutation onward and they are called frameshift mutations
 Frameshift causes two problems
1) All codons from the frameshift on will be
different and most likely yield a useless
protein
2) Stop signal are misread and so there may either be
premature stoppage of translation or translation may
proceed beyond the gene
Figure 3. A frameshift mutation showing possible effects of
insertion of a single base resulting in the creation of a new
stop sequence on the mRNA
Back Mutation and suppression
 A second point mutation on the same gene can have three
effects
– Mutation can result in another mutant codon or one codon
that has experienced two changes (double mutation)
– If mutation is at the same site, the original sequence can be
returned, an effect known as back mutation
– Intragenic suppression can occur and this occurs when a
second mutation in the same gene masks the occurrence of
the original mutation without actually restoring the original
sequence. The new sequence is a double mutation but with
the same phenotype
 Suppressed mutations can be distinguished from back
mutations through genetic crosses and DNA sequencing
Conditional lethality
 These are mutants that are lethal under certain conditions but
not under others
 Nutritional-requirement mutants reflect failure of one or more
enzymes in the biosynthetic pathways of the bacteria
 Temperature-sensitive mutants are another class they are
normal at 25 oC (permissive temperature) but cannot make
DNA at 42 oC (restrictive temperature)
Spontaneous mutagenesis
 If the base of a DNA underwent a proton shift into one of its
rare tautomeric forms (tautomeric shift) during replication, an
inappropriate pairing of bases would occur
 If a purine (or pyrimidine) is replaced by another purine (or
pyrimidine) through a translational state involving a
tautomeric shift this is referred to as transition mutation
 The form of replacement in which purine replaces a
pyrimidine or vice versa is referred to as transversion
mutation.
Chemical mutagenesis
 Mutation can be caused by (a) Radiation (b) Chemicals (3)
Temperature (4) Enzymatic errors and (5) Spontaneous decay
 Mice or rats are used to determine if cexpensive and time
consuming
 Ames test was introduced to find out whether new chemicals are
mutagenic. S. typhimurium requires histidine and so will not
growth in a minimal medium. However, the strain will grow in
minimal medium if a mutagen is present because it causes
histidine pathway to revert to wild type. So if an agent is
mutagenic S. typhimurium will grow in minimal medium
Chemical mutagenesis
 Translations
Translations are routinely produced by base analogues. Two examples of these
analogues are pyrimidine analogue 5-bromouracil (5BU) and purine analogue 2aminopurine (2AP). Both act the same and target the base thymine. They also
targets guanine
 Transversions
Ethyl methane sulfonate and ethyl ethane sulfonate causes removal of the
purine ring and DNA polymerase III will be free to insert any of the four bases. If
thymine is placed the original base pair is restored, insertion of cytosine will
result in transition while insertion of adenine or guanine will result in transversion
 Insertions and deletions
These are caused by molecules of acridine dyes, such as proflavin and acridine
orange
 Misalignment mutagenesis
Additions or deletions in DNA can occur by misalignment of a template strand
and the newly (progeny) formed strand in a region in which there is a repeated
sequence.
Intergenic suppression
 When mutation occurs, back mutation or intragenic
suppression will lead to the survival of the individual
 A third route is intergenic suppression which is the restoration
of the function of a mutated gene by changes in a different
gene called, suppressor gene
 Suppressor genes are usually tRNA and when mutated they
change the way in which a codon is read
DNA repair
 Thousands of bases of DNA are damaged each day. Cells have
evolved mechanisms to repair and these mechanisms are
placed in three broad categories
(1) Damage reversal
(2) Excision repair
(3) Postreplicative repair
Damage reversal
 In E. coli DNA photolyase normally binds in the dark to create
thymine-thymine dimmers. When light shines this enzyme will
break the dimmer bonds with light energy thus reversing
dimerization
Excision repair
 This is the repair in which damaged bases are removed and
patched. There are three types of this repair
(a) UV damage repair
(b) AP repair
(c) mismatch repair
Excision repair
UV damage repair
 When DNA is exposed to UV dimmers are formed. An
endonuclease enzyme known as ABC exinuclease will
hydrolyze the damaged strand DNA helicase II will separate
the strands and DNA polymerase I and ligase will fill in the
gap
Excision repair
AP repair

This is the repair of the apurinic and a pyrimidinic sites on
DNA. These are sites in which a base has been removed
by radiation or DNA glycosylases (enzyme that sense
damaged DNA and remove it). AP endonucleases will
nick the DNA. An exonuclease will remove the short
length of DNA and DNA polymerase I and ligase will
repair the patch
Excision repair
Mismatch repair
 This is responsible for 99% of all repairs to DNA.
 As DNA polymerase replicates DNA some errors occur that are
not corrected by the proof reading
 .These errors are corrected by the mismatch repair system whose
members are specified by mutatorH, mutatorL, mutatorS and
mutatorU genes
 This system works such that the mismatch is recognized by the
products of the mutatorL and mutatorS genes. The product of
mutatorH nicks the DNA. While the product of mutatorU gene
(also called uvrD), DNA helicase II unwinds the nicked region so
it falls free. DNA polymerase I and ligase will then patch up
Postreplicative repair
 When E. coli DNA polymerase encounters some damages,
such as thymine dimmers, it stops, skips down the DNA,
leaving a gap before starting replication
 Repairing of this gap is done by a group of enzymes at the
recA locus
 Since repair occurs due to failure of replication the process is
called postreplication failure it is also known as
recombinational repair
Recombination
 This is a breakage and reunion process. Homologous parts of
chromosome come into apposition, at which point both strands
are broken and then reconnected in a crosswise fashion
Figure 4. Crossover of homologous chromosomes during
meiosis between the loci
Holliday mechanism of breakage and repair
reunion

Two homologous DNA will form a duplex

Breakage of the duplex, each at the same place and
only on one strand of each duplex

The broken strands then pair with the complement on
the other duplex and covalent bonds are formed

Branch migration occurs

In order to release cross-linked duplexes, a second cut
and it occurs in the previously uncut strand
Figure 5. The Holiday model of genetic recombination