DNA Damage and Repair
An Overview
• Because each cell contains only one or two copies of its DNA, the
DNA sequence is highly protected from harm.
• DNA is a relatively stable molecule, but Earth’s natural environment
is quite toxic, and damage to DNA is inevitable.
• DNA can also be altered by mistakes made during its own replication
or recombination.
• Damage and sequence alterations to DNA are often quickly repaired,
but when they are not, the DNA becomes permanently altered and
harbors a mutation.
• Mutations are changes in DNA sequence, and when mutations occur
in germ-line cells, these changes are inheritable.
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• A cancer cell has mutations that prevent cell death, resulting in loss of
cell cycle control and unregulated cell division, which leads to
malignant tumors that can end the life of the entire organism.
• The cell has a limited amount of time to fix the initial alteration and
restore the DNA to its normal sequence, before replication converts
the alteration into a mutation that will be passed on to the next
generation.
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•
•
Mutation
Somatic mutations : occur in somatic cells and only affect the individual in
which the mutation arises.
Germ-line mutations: alter gametes and passed to the next generation.
• Mutations are quantified in two ways:
1. Mutation rate = probability of a particular type of mutation per unit time
(or generation).
2. Mutation frequency = number of times a particular mutation occurs in a
population of cells or individuals.
Types of DNA mutations
• A mutation is a change in a DNA sequence that is propagated through
cellular generations. Mutations can be as small as a single base pair or
can range from a few base pairs to thousands.
• Mutations of one or a few base pairs usually result from errors in
replication or damaged nucleotides.
•
Mutation can have different effects on gene function.
A mutation in a gene product can result in a loss of
function or a gain of function
loss of
function
e.g. Truncated protein,
disrupt the regulation of
gene expression.
Gain of
function
e.g. Increase the affinity of
an enzyme for its substrate,
up-regulate gene expression.
Studies in Drosophila melanogaster suggest that mutations that alter
the protein sequence are most likely to be harmful; about 70% have a
negative effect, and the rest either are neutral or have a weak
beneficial effect.
A Point Mutation Can Alter One Amino
Acid
• A change in a single base pair is often
referred to as a point mutation.
• Point mutations fall into two
categories:
i.
ii.
A transition mutation is the exchange
of a purine-pyrimidine base pair for the
other purine-pyrimidine base pair: C≡G
becomesT=A, or T=A becomes C≡G.
A transversion mutation is the
replacement of a purine-pyrimidine base
pair with a pyrimidine-purine base pair,
or vice versa.
For example, C≡G becomes either G≡C
or A=T.
• Transition mutations are nearly 10 times more frequent than
transversions.
• A point mutation in the protein-coding region of a gene can result
in an altered protein with partial or complete loss of function.
• If the protein is central to cell viability, the cell could die.
• Point mutations are known to cause a wide variety of human diseases.
One example is sickle-cell anemia, resulting from a transversion that
produces an amino acid change in hemoglobin: a GAG encoding
glutamate (E) at residue 6 changes to GTG, encoding valine (V)
(E6V).
But: such type of mutation can considered as harmful
type of mutation?
• The most harmful mutations are those occurring in the genes involved
in DNA repair, because these often result in cancer.
• Many mutations in one cell can result in cancer because a mutation
will occur in a gene (or genes) that encodes a protein needed to
control cell division.
• In normal cells, oncogenes encode proteins that drive the cell
division cycle forward, and tumor suppressor genes encode
proteins that suppress cell division.
• Many tumor suppressors are transcription factors that regulate the
expression of genes that drive the cell cycle.
• The transcription factor p53 and the retinoblastoma protein are
examples of tumor suppressors that are mutated in many types of
cancer.
Point mutations in a protein-coding region can be
classified by their effect on the protein sequence.
• The DNA sequence encoding a protein is read in codons. Each codon
corresponds to an amino acid .
• A silent mutation is a nucleotide change that produces a codon for
the same amino acid. For example, GAA and GAG both code for
glutamate.
• A missense mutation is a nucleotide change that results in a
different amino acid, such as a change from glutamate (GAA) to
glutamine (CAA).
• A nonsense mutation changes the nucleotide sequence so that
instead of encoding an amino acid, the triplet functions as a stop
codon, terminating the protein.
Small Insertion and Deletion Mutations
Change Protein Length
• Another type of mutation is the gain or loss of one or
more base pairs.
i.
Insertion mutations occur when one or more base
pairs are added to the wild-type sequence.
i.
Deletion mutations are due to the loss of one or more
base pairs.
•
Insertion and deletion mutations are collectively
referred to as indels.
The DNA sequence from the start codon to the stop
codon is referred to as a reading frame.
•
• Because nucleotides are decoded in triplets, an indel mutation of only
one or two base pairs in the coding sequence of a protein throws off
the reading frame after the mutation, resulting in a frameshift
mutation.
Types of mutations in ORFs
1- Nonsynonymous/missense mutation
•
Base pair substitution results in substitution of a different amino acid.
2- Nonsense mutation
•
Base pair substitution results in a stop codon (and shorter polypeptide).
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3- Neutral nonsynonymous mutation
• Base pair substitution results in substitution of an amino acid with
similar chemical properties (protein function is not altered).
4- Synonymous/silent mutation
• Base pair substitution results in the same amino acid.
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5- Frameshift mutations:
• Deletions or insertions (not divisible by 3) result in translation of
incorrect amino acids, stops codons (shorter polypeptides), or readthrough of stop codons (longer polypeptides).
Mechanisms of DNA Repair
Can you considered the
mistakes
made
during
replication
and
recombination that do not
involve damaged bases as
mutations?
Mismatch Repair Fixes Misplaced-Nucleotide
Replication Errors
• Mismatched nucleotides incorporated by the replication apparatus are
corrected by the mismatch repair (MMR) system, which is conserved in
all cell types from bacteria to humans.
• The mismatches are nearly always corrected to reflect the information in the
parent strand.
• the E. coli MMR system can also recognize small loops of up to 4 bp of
unpaired nucleotides.
• Left unrepaired, these small loops of extra DNA result in deletions or
insertions.
• Loops of more than 4 bp are not recognized by the MMR system. Thus,
larger indels are simply not corrected.
Mechanisms of DNA Repair
• The most prevalent means that cells use to repair damaged DNA is
excision repair, of which there are two types: base excision and
nucleotide excision repair.
1. Base excision repair (BER) functions at the level of a single damaged
nucleotide that distorts DNA very little. It is also the main pathway for the
repair of single-strand DNA breaks that lack a ligatable junction and
therefore require “cleaning” of the 3′ or 5′ terminus for ligation.
2.
Nucleotide excision repair (NER) targets large, bulky lesions and
removes DNA on either side of them. In contrast to base excision repair,
NER does not require specific recognition of a damaged nucleotide and
thus it can remove DNA lesions.
Base excision repair.
(a) In bacteria, a glycosylase excises a
damaged nucleotide base, then an AP
endonuclease nicks the backbone at the
abasic site. Nick translation by Pol I
excises the 5′ deoxyribose phosphate (5′dRP) and some dNMPs, and synthesizes a
new strand. Ligase seals the gap.
(b) Eukaryotic BER, after the first two
steps, can take either of two paths. In long
patch repair, a DNA polymerase extends
the DNA strand from the 3′ terminus,
displacing the 5′ single-stranded DNA; this
is followed by cleavage by a flap
endonuclease and ligation. In short patch
repair, only one nucleotide is inserted (by
Pol β) prior to ligation.
The NER pathway uses several
proteins, including UvrA, UvrB and
UvrC, that recognize the lesion and
make incisions on either side,
allowing UvrD (helicase II) to
displace a section of lesioncontaining DNA. The single-strand
gap is filled in by Pol I, and the
DNA is sealed by ligase. A
transcription coupled repair (TCR)
path can also be taken in which
RNA polymerase stalls at the lesion
on the coding strand. After the
RNA polymerase is displaced, the
reaction proceeds as shown here,
using UvrA through UvrD, Pol I,
and ligase.