MUTATIONS AND DNA REPAIR

-In molecular biology and genetics, mutations are changes in a

genomic sequence, and can be defined as sudden and spontaneous
changes in the cell.
- Mutations are caused by radiation, viruses and mutagenic chemicals,
as well as errors that occur during meiosis or DNA replication, also can
be induced by the organism itself, by cellular processes such as
hypermutation.
-Mutation can result in several different types of change in sequences;
(DNA) these can either have no effect, alter the product of a gene, or
prevent the gene from functioning properly or completely.
-Due to the damaging effects that mutations can have on genes,
organisms have mechanisms such as DNA repair to remove mutations.
-Viruses that use RNA as their genetic material have rapid mutation
rates, which can be an advantage since these viruses will evolve
constantly and rapidly, and thus evade the defensive responses of e.g.
the human immune system.
-Mutations can involve large sections of DNA becoming duplicated,
usually through genetic recombination.
Causes of mutations
- Two classes of mutations are spontaneous mutations and induced
mutations caused by mutagens.
Spontaneous mutations can be caused by:
1- Tautomerism: A base is changed by the repositioning of a hydrogen
atom, altering the hydrogen bonding pattern of that base resulting in
incorrect base pairing during replication.
2- Depurination: Loss of a purine base (A or G) to form an apurinic site
(AP site).
3- Deamination: changes a normal base to an atypical base containing a
keto group in place of the original amine group.
4- Slipped strand mispairing: Denaturation of the new strand from the
template during replication, followed by renaturation in a different spot
("slipping"), which can lead to insertions or deletions.
Induced mutations can be caused by:
A- Chemicals
-Hydroxylamine
-Alkylating agents (e.g. nitrosourea) which can mutate both replicating
and non-replicating DNA.
-In contrast, a base analog can only mutate the DNA when the analog is
incorporated in replicating the DNA.
-DNA intercalating agents (e.g. ethidium bromide)
-DNA crosslinkers
-Oxidative damage
- Nitrous acid converts amine groups on A and C to diazo groups,
altering their hydrogen bonding patterns which leads to incorrect base
pairing during replication.
B- Radiation
-Ultraviolet radiation (nonionizing radiation).
-Two nucleotide bases in DNA – cytosine and thymine – are most
vulnerable to radiation that can change their properties.
-UV light can induce adjacent pyrimidine bases in a DNA strand to
become covalently joined as a pyrimidine dimer.
-UV radiation, particularly longer-wave UVA, can also cause oxidative
damage to DNA.
-Ionizing radiation
-Radioactive decay, such as 14C in DNA
C- Viral infections
Classification of mutation types
By effect on structure
-The sequence of a gene can be altered in a number of ways.
-Mutations in the structure of genes can be classified as:
A- Small-scale mutations, such as those affecting a small gene in one or
a few nucleotides, including:
1- Point mutations, often caused by chemicals or malfunction of DNA
replication, exchange a single nucleotide for another.
- These changes are classified as transitions or transversions.
- Most common is the transition that exchanges a purine for a purine (A
↔ G) or a pyrimidine for a pyrimidine, (C ↔ T).
Less common is a transversion, which exchanges a purine for a
pyrimidine or a pyrimidine for a purine (C/T ↔ A/G).
-A point mutation can be reversed by another point mutation, in which
the nucleotide is changed back to its original state (true reversion) or by
second-site reversion.
- Point mutations that occur within the protein coding region of a gene
may be classified into three kinds, depending upon what the erroneous
codon codes for:
1- Silent mutations: which code for the same amino acid.
2- Missense mutations: which code for a different amino acid.
3-Nonsense mutations: which code for a stop and can truncate the
protein.
2- Insertions add one or more extra nucleotides into the DNA.
-They are usually caused by transposable elements, or errors during
replication of repeating elements (e.g. AT repeats).
-Insertions in the coding region of a gene may alter splicing of the
mRNA (splice site mutation), or cause a shift in the reading frame
(frameshift), both of which can significantly alter the gene product.
- Insertions can be reverted by excision of the transposable element.

3- Deletions remove one or more nucleotides from the DNA.

-Like insertions, these mutations can alter the reading frame of the gene.
- They are generally irreversible: though exactly the same sequence
might theoretically be restored by an insertion, transposable elements
able to revert a very short deletion (say 1–2 bases) in any location are
either highly unlikely to exist or do not exist at all.
-Note that a deletion is not the exact opposite of an insertion: the former
is quite random while the latter consists of a specific sequence inserting
at locations that are not entirely random or even quite narrowly defined.
B- Large-scale mutations in chromosomal structure, including:
1- Amplifications (or gene duplications) leading to multiple copies of all
chromosomal regions.
2- Deletions of large chromosomal regions, leading to loss of the genes
within those regions.
3- Mutations potentially bringing together separate genes to form
functionally distinct fusion genes (e.g. bcr-abl). These include:
I- Chromosomal translocations: interchange of genetic parts from
nonhomologous chromosomes.
II- Interstitial deletions: an intra-chromosomal deletion that removes a
segment of DNA from a single chromosome, thereby apposing
previously distant genes.
III- Chromosomal inversions: reversing the orientation of a
chromosomal segment.
VI- Loss of heterozygosity: loss of one allele, either by a deletion or
recombination event, in an organism that previously had two different
alleles.
By inheritance
- By pattern of inheritance The human genome contains two copies of
each gene – a paternal and a maternal allele.
1- A heterozygous mutation is a mutation of only one allele.
2- A homozygous mutation is an identical mutation of both the paternal
and maternal alleles.
3- Compound heterozygous mutations or a genetic compound
comprises two different mutations in the paternal and maternal alleles.
4- A wild type or homozygous non-mutated organism is one in which
neither allele is mutated. (Just not a mutation).
Special classes
Conditional mutation
For example, a temperature-sensitive mutation can cause cell death at
high temperature (restrictive condition), but might have no deleterious
consequences at a lower temperature (permissive condition).

DNA repair systems

-Repair mechanisms are divided into 3 categories:
1- damage reversal simplest; enzymatic action restores normal structure
without breaking backbone
2- damage removal involves cutting out and replacing a damaged or
inappropriate base or section of nucleotides.
1- Damage reversal
A- Photoreactivation
- This is one of the simplest and perhaps oldest repair systems: it consists
of a single enzyme which can split pyrimidine dimers (break the
covalent bond) in presence of light.
-The photolyase enzyme catalyzes this reaction; it is found in many
bacteria, lower eukaryotes, insects, and plants.
-It seems to be absent in mammals (including humans).

B- Ligation of single strand breaks

-X-rays and some chemicals like peroxides can cause breaks in backbone
of DNA.
-Simple breaks in one strand are rapidly repaired by DNA ligase.
- Microbial mutants lacking ligase tend to have high levels of
recombination since DNA ends are recombinogenic (very reactive).
2- Damage removal
A- Base excision repair
-The damaged or inappropriate base is removed from its sugar linkage
and replaced.
-These are glycosylase enzymes which cut the base-sugar bond as uracil
glycosylase enzyme which removes uracil from DNA to provide AP site.
- Uracil is not supposed to be in DNA.
-It can occur if RNA primers not removed in DNA replication or
(more likely) if cytosine is deaminated (this is potentially mutagenic).
-The enzyme recognizes uracil and cuts the glyscosyl linkage to
deoxyribose.
-Then, the AP endonuclease removes the AP site and neighboring
nucleotides.
-The gap is filled by DNA polymerase I and DNA ligase using the other
strand as a template.
B- Mismatch repair
-The repairing process begins with the protein MutS which binds to mismatched base
pairs.
-Then, MutL is recruited to the complex and activates MutH which binds to GATC
sequences.
-Activated MutH cleaves the unmethylated strand at the GATC site.
-Subsequently, the segment from the cleavage site to the mismatch is removed by
exonuclease (with assistance from helicase II and SSB proteins).
-If the cleavage occurs on the 3' side of the mismatch, this step is carried out by
exonuclease I (which degrades a single strand only in the 3' to 5' direction).
-If the cleavage occurs on the 5' side of the mismatch, exonuclease VII.
-The gap is filled by DNA polymerase III and DNA ligase.
The distance between the GATC site and the mismatch could be as long as 1,000 base
pairs. Therefore, mismatch repair is very expensive and inefficient.
-Mismatch repair in eukaryotes may be similar to that in E. coli. Homologs of MutS
and MutL have been identified in yeast, mammals, and other eukaryotes.
-MSH1 to MSH5 are homologous to MutS; MLH1, PMS1 and PMS2 are homologous
to MutL.
-Mutations of MSH2, PMS1 and PMS2 are related to colon cancer.
- In eukaryotes, the mechanism to distinguish the template strand from the new strand is
still unclear.
C- Nucleotide excision repair (NER)
-This system works on DNA damage which is "bulky" and creates a
block to DNA replication and transcription (so, UV-induced dimers and
some kinds of chemical adducts).
-In E. coli, proteins UvrA, UvrB, and UvrC are involved in removing
the damaged nucleotides.
-The gap is then filled by DNA polymerase I and DNA ligase.
- In yeast, the proteins similar to Uvr's are named RADxx ("RAD"
stands for "radiation"), such as RAD3, RAD10.
-Humans with the hereditary disease Xeroderma pigmentosum are
sunlight-sensitive, they have very high risks of skin cancers on sun-
exposed areas of the body and have defects in genes homologous to
those required for NER in simple eukaryotes.
-NER mutants in lower organisms are UV-sensitive.