Lecture Outlines:

 Explain the types, molecular causes of

DNA mutations.
 Explain the biochemical consequences of
DNA mutations.
 Explain the molecular mechanisms and
enzymes that repair DNA damage.
 Compare different DNA repair pathways.
 Identify examples of diseases associated
with defective DNA repair mechanisms.
 Mutation and DNA damage
 Definition of Mutation:
Permanent changes in DNA sequences
 Causes of Mutation and DNA damage:
1-error in replication:
By pass proofreading and repair system
2-spontaneous changes:
A-Spontaneous depurination or depyrimidination
B-Spontaneous Deamination:
Three bases (A, G, C) of DNA contain amino groups,
A to Hypoxanthine, G to Xanthine and C to Uracil.
1. Depurination: A, G
2. Deamination: C --> U
 Base Deamination
• Nucleotide bases can undergo
spontaneous loss of their
exocyclic amino groups
(deamination).
• Under typical cellular conditions,
deamination of cytosine in DNA to
uracil occurs in about one of every
107 residues every 24 hours.
• A and G deamination occurs at
1/10 of this rate.

06.2.27 5
3-Chemicals:
A-Intercalating agents:
such as ethidium bromide and actinomycin D inhibit
binding of DNA-binding proteins such as DNA
polymerases, RNA polymerases, topoisomerases, and
other enzymes and cause inseration or deletion
mutation of one or more nucleotide(s).
B-incorporation of base analogue
C-Nitrous acid (HNO2):
Causes deamination of bases
4-irradiation:
A-Ultraviolet light:
induced thymine-thymine (pyrimidine) dimer

B-ionizing radiation: (X-ray and gamma rays)

Cause double strand breaks and depurination
Pyrimidine dimers (UV induced).
 The genetic code
 To crack the genetic code found in
DNA we need to look at the sequence
of bases.

 The bases are arranged in triplets called

codons.
 The genetic code
 The correspondence between the sequence
of bases in the mRNA nucleotides and the
sequence of amino acids in the protein
synthesized is called the “genetic code”.

 Each 3 successive bases in the coding region

of the mRNA form a “codon”, which
correspond to a specific amino acid.
 The genetic code
 In a codon, since each position of the 3 may be occupied
by one of four bases (uracil, cytosine, adenine, or
guanine), there are 43 = 64 possible codons.
 Actually, 61 of these codons code for the 20 amino acids
used for protein synthesis.
 The remaining 3 codons (UAA, UAG and UGA) indicate
termination of the peptide chain, i.e. stop codons.
 One codon, AUG, in addition to coding for methionine,
also codes for initiation of protein synthesis, i.e.,
initiator codon.
 The sequence of nucleotides in a codon is always written
in the direction 5’ to 3’.
 The Characteristics of genetic code
A. Specificity
The genetic code is specific (unambiguous); this means
that a certain codon always codes for only one specific
amino acid . For example: the codon UUU always code
for phenylalanine.

B. Universality
With minor exceptions in the mitochondria, the same
genetic code applies to all living organisms.
 The Characteristics of genetic code
C.Redundancy or degeneracy
A given amino acid may have more than one codon; e.g.,
leucine has 6 codons.
Only methionine and tryptophan has one codon each.
The third base in codon is the least important; e.g., glycine
has the codons GGU, GGC, GGA, and GGG.

D. Reading frame (non-overlapping and commaless):

The codons are read from a fixed specific starting point
(initiating codon) on the mRNA as a continuous
uninterrupted sequence of bases taken 3 at a time.
Small Scale mutation (DNA sequence mutation):
A-point mutation:
1- Transition mutation
2- Transversion mutation
B- mutation causing change in the amount or
structure of the protein produced by translation :
I. Insertion or deletion or duplication of the
multiple of 3 nucleotides
II. Insertion or deletion or duplication of not the
multiple of 3 nucleotides
III. Splice site mutations
IV. Dynamic/unstable Trinucleotide repeat
expansion
 point mutation :
Changing one nucleotide (base substitution,
single base mutation, maybe :

1- Transition mutation:
where a purine is changed into another purine or a
pyrimidine is changed into another pyrimidine.

2- Transversion mutation:
where a purine is changed into a pyrimidine, or
a pyrimidine is changed into a purine.
The effect of Single base substitutions mutation
may be:
1. Silent Mutation
.

 This occurs if the resulting codon still codes for the same amino acid.
 It is more likely to occur if the change occurs in the third base of the
codon.
 If A in the codon for serine, UCA, is changed to C, the resulting
codon, UCC, still codes for serine.
2. Missense Mutation
 This occurs if the resulting codon codes for a different amino acid.
 It is more likely to occur if the change occurs in the first or second
base of the codon.
 If U in the codon for serine, UCA, is changed to C, the resulting
codon, CCA, codes for proline.
3. Nonsense Mutation
 This occurs if the resulting codon codes for termination of the
peptide chain.
 If C in the codon of serine, UCA, is changed to G, the resulting codon,
UGA, leads to termination of translation.
The effect of base pair substitutions:
Mis-sense Mutation of Sickle cell anemia
The effect of base pair substitutions and other mutations:
The effect of other mutation may be:
I- Addition or Deletion of One or Two Nucleotides
(not the multiple of 3 nucleotides)
This results in a frame shift mutation, leading to a
change in all codons after the addition or the deletion.

II- Addition or Deletion of 3 Nucleotides

(the multiple of 3 nucleotides)
This leads to addition or deletion of one amino acid(s)
to the peptide chain. The reading frame is not
changed.
The effect of base pair substitutions and other mutations:
The effect of other mutations:
The effect of other mutation may be:
III. Splice site mutations:
Mutations at splice sites can alter the way in which introns
are removed from pre-mRNA molecules, producing
aberrant proteins.
The effect of other mutation may be:
III. Splice site mutations:
The effect of other mutation may be:
IV. Dynamic/unstable Trinucleotide repeat expansion:
 Occasionally, a sequence of three bases that is repeated in
tandem will become amplified in number, so that too many
copies of the triplet occur.

 If this occurs within the coding region of a gene, the protein

will contain many extra copies of one amino acid.

 If the trinucleotide repeat expansion occurs in the

untranslated portion of a gene, the result can be a decrease
in the amount of protein produced.

 As Fragile X Syndrome that occurs due to expansion of

trinucleotide repeats (CGG)n at the X chromosome.
 DNA Repair phases:

1. Recognition of the damage (lesion) on the

DNA
2. Removal or excision of the damage
3. Replacement or filling the gap left by excision
using the sister strand as a template for DNA
synthesis
4. Ligation.
1-Mismatch repair(MMR)(repair of mismatched bases)
 Sometimes replication errors escape the proofreading function
during DNA synthesis, causing a mismatch of one to several
bases.
 In E. coli, mismatch repair is mediated by a group of proteins
known as the Mut proteins. Analogous proteins are present in
humans.
 In bacteria, mismatches of bases in newly synthesized DNA
strands are recognized by a repair system, since parental
template strands of DNA are methylated.
 Newly synthesized DNA strands are not methylated
immediately after DNA synthesis, and therefore, a mismatched
base on the unmethylated DNA strand is known.
1-Mismatch repair(MMR)(repair of mismatched bases)
 Three key components are required for mismatch repair
systems in E. coli: MutS, MutL, and MutH proteins.
 If a TG mismatch was introduced in DNA, MutS protein
scans the DNA sequence and binds to the mismatched
base.
 Then MutH protein, which is an endonuclease, recognizes
the methylated DNA sequence of GATC on the parental
strand,
 MutL links MutH and MutS.
 The linked MutH protein nicks the opposite strand of the
methylated A base on the parental strand. Then, the
helicase, UvrD, unwinds DNA from the nick and proceeds
past the mismatched nucleotide.
1-Mismatch repair(MMR)(repair of mismatched bases)

 Exonucleases cut away the nicked GATC

sequence, and single-strand binding proteins
stabilize the resulting single strand.
 The excised DNA region is resynthesized by
DNA polymerase III.
 DNA ligase seals the nick to form double-
stranded DNA.
 The end result of the mismatch repair system
is a TG mismatch correction corresponding to
the methylated parental strand sequence.
1-Mismatch repair(MMR)(repair of mismatched bases)

 In eukaryotes, a DNA single base mismatch is

recognized by MSH2-MSH6 heterodimer
proteins, and MLH1, PMS2, and EXO1 proteins
serve the function of MutL in prokaryotes.

 However, the counterpart of prokaryotic MutH

protein has not been identified in eukaryotes.
1-Mismatch repair(MMR)(repair of mismatched bases)
 Hereditary nonpolyposis colon cancer
(HNPCC) is caused by mutations of one of five
genes: hMSH2, hPMS1, MSH6, hMLH1, and
hPMS2.
 These gene products are involved in the DNA
mismatch repair system, and defects in any of
these DNA repair genes predispose individuals
to colon cancer, as well as to other cancers, due
to microsatellite instability, which is a hallmark
of HNPCC.
1-Mismatch repair(MMR)(repair of mismatched bases)
2-Base excision repair (BER)
 It handles a wide variety of individual base
damage caused by oxidation and
deamination.
 Three major steps are involved in BER:
1- the damaged base is recognized and removed
by an appropriate DNA N-glycosylase. The DNA
N-glycosylases remove the damaged bases out
of the DNA strand by cutting glycosidic
bonds between deoxyribose sugar and
heterocyclic bases, creating an apurinic or
apyrimidinic (AP) site.
2- another enzyme called AP endonuclease
creates a nick by cleaving the sugar-
phosphate backbone at the AP site, creating a
3’-OH terminus adjacent to the AP site.
3- The gap at the AP site is then filled by the
action of DNA polymerase and DNA ligase.
3-Nucleotide excision repair (NER)
 is effective in removing bulky DNA damage
caused by UV light, oxidative chemicals,
reactive oxygen species, cross-linking agents,
and intercalating drugs.
 The E. coli NER system requires four proteins:
UvrA, B, C, and D.
 UvrA dimer recognizes DNA damage and binds
to the damaged site with UvrB.
3-Nucleotide excision repair (NER)
 Then the UvrA dimer is replaced by UvrC to
form a UvrBC complex.
 The UvrBC complex cleaves a 5th phosphodiester
bond at the 3’ side and an 8th phosphodiester
bond at the 5’side of the damaged DNA site.
 Then UvrD (helicase) unwinds the DNA to
release the damaged DNA strand and expose the
single-stranded region.
 DNA polymerase I fills the excised regions, and
DNA ligase seals the nick, completing the repair
process
3-Nucleotide excision repair (NER)
 the human NER system includes 7 proteins,
which collaborate to repair DNA damage.
 The human disease xeroderma pigmentosum
(XP) is caused by defects in the NER system.
 Investigations of XP patients have led scientists
to identify the NER components: XPA to XPG.
 In eukaryotes, XPA protein binds to the
damaged DNA site.
 XPB and XPD proteins are helicases to unwind
the damaged DNA duplex.
3-Nucleotide excision repair (NER)
 XPG cleaves the 3’ side of the damaged strand,
and the XPF/ERCC1 complex cleaves the 5’ side
of the damaged strand, generating a single-
stranded region of 24 to 32 bases covering the
damaged site.

 The single-stranded portion is filled with

correct base sequences by DNA polymerase δ/ε
along with regular DNA replication complexes.
4-DNA double-strand breaks repair:
DNA double-strand breaks are
repaired by two main mechanisms:

A-homologous recombination (HR).

B- nonhomologous end joining (NHEJ).

4-DNA double-strand breaks repair:
A-homologous recombination (HR)
 The HR repair system is more accurate than the
NHEJ system, since it uses the homologous DNA
sequence on the sister chromatid to repair the
damaged region.
 In HR repair, Rad51 protein searches the
homologous copy of the damaged DNA on the
sister chromatid, and the DNA damage is
repaired by copying the DNA sequence from the
sister chromatid.
4-DNA double-strand breaks repair:
B- nonhomologous end joining (NHEJ):
 the NHEJ repair system does not require an identical
copy of the DNA sequence to repair the DNA damage.

 Instead, the system joins two ends of the broken DNA

double helixes by DNA ligase IV/Xrcc4 complex.

 First, Ku70/80 heterodimer protein recognizes double-

strand breaks, has latent ATP-dependent helicase
activity and recruits the protein kinase DNA-dependent
PKcs.
4-DNA double-strand breaks repair:
B- nonhomologous end joining (NHEJ):(Cont.)

 The DNA-dependentPKcs autophosphorylate themselves to

prevent possible nuclease attack and also to promote
ligase reactions.

 compared to HR, NHEJ is a much more error-prone

repair mechanism.