A short presentation on DNA damage/mutation and the repair mechanisms involved that I prepared for my online chapter presentation. (The content and pictures credited to Watson et al.'s Molecular Biology of the Gene, 7th Edition)

1. The Mutability
and Repair of
DNA
Sanam Parajuli
Content and Pictures from: Molecular Biology of the
Gene(7th Edition), Watson et al.

2. Introduction
• With every division, a living cell has to copy billions of its base pairs to a
new DNA strand, so, mistakes are unavoidable
• During normal functioning too, a living organism requires proper
expression and functioning of thousands of genes
• Mutations: possible in coding sequences or in flanking sequences
governing gene expression
• Very high rates of mutation in the soma can lead catastrophic incidences of
cancer; too much changes in the germline can lead in progeny not surviving
• But too little changes: no evolution, no speciation, no diversity
• Life and biodiversity: sweet balance between DNA damage and repair

3. Intro…
• Two sources of mutation: Inaccuracy in DNA replication and Chemical Damage to
the genetic material
• Replication errors: because of tautomerization that leads to incorrect pair
readings and although the enzymatic machinery of DNA replication attempts to
correct these errors, some escape detection
• Tautomerization: rearrangement of atoms in a molecule, eg: Adenine and
cytosine from amino to imino form, guanine and thymine from keto to enol form.
Now, A-C pairs and G-T pairs.
• DNA damage: because of finite stability, DNA is subject to damage such as loss of
bases, chemical alteration of bases because of environmental mutagens
• Transposons: Jumping genes (not dealt in this chapter)

5. Consequences of replication errors and DNA
Damage
• 1. Introduction of permanent changes to the DNA (mutations), which can alter
the coding sequence of a gene or its regulatory sequences, effects observed in
the progeny
• 2. Changes to the DNA can prevent it from being used as a template for
replication and transcription, immediate effects observed in the cell itself in
function and survival
• Challenge for a cell: scan the genome for errors and also mend the errors if
possible
• How is the DNA mended rapidly enough to prevent setting of mutations? How
are parent and daughter strands distinguished in repair? How does the cell
restore the proper DNA sequence when, because of a break or severe lesion, the
original sequence can no longer be read? How does the cell cope with lesions
that block replication?
• It depends on the type of error to be repaired.

6. Replication Errors and their Repair
The Nature of Mutations
• Simplest kinds: switches of one base for another:
Transitions and Transversions
• Point mutations: alter a single nucleotide
• Other kinds like extensive insertions and deletions can
cause more drastic changes to the DNA
• Ex: insertion of a transposon; rearrangement of
chromosomes; aberrant recombination events
• Probability that new mutations arise spontaneously at any
given site on the chromosome: 10-6 to 10-11 per round of
DNA replication
• Some chromosome sites can be “hotspots” for mutations
while some can mutate less frequently

7. *DNA Microsatellites*
• repeats of simple di-, tri-, or tetranucleotide sequences
• Really prone to mutations
• Ex: CA stretches found scattered across chromosomes
• DNA replication at such sites prone to “slippage” and a population, thus,
can have polymorphic CA stretches at a given point
• Such polymorphism used as physical marker for mapping inherited
mutations
• Triple repeat expansions associated diseases: Fragile X (CGG repeats more
than 200), Huntington’s (CAG repeats coding for excess glutamine) etc

8. Some replication errors escape proofreading
• The 3’5’ exonuclease in the DNA polymerase: removes wrongly incorporated
nucleotides with high accuracy. This proofreading increases the fidelity of DNA
replication by a factor of nearly 100
• However, not foolproof
• Some misincorporated nucleotides escape detection and become a mismatch
between the template and new strand
• For each nucleotide, there are 3 possible mismatches (eg: for T, T:C, T:G, and T:T
are mismatches). So, there are 12 possible mismatches for the 4 nucleotides
• If misincorporated nucleotides not detected and corrected, the sequence change
will be permanent in the genome

10. Mismatch Repair System Removes Errors that
Escape Proofreading
• Fortunately, a mismatch repair system exists for correcting errors that
escape exonuclease repairing that increases the replication accuracy
by an additional 2 or 3 orders of magnitude
• Challenge for this system: First: scanning for mismatches. Mismatches
are transient, only true for the cell of origin, in the daughter cell, it
becomes permanent
• Second: only the daughter strand needs repair, not the parent. How
does the system know which strand to repair?

11. Mismatch Repair System in E. coli
• In E. coli, mismatches are repaired by the dimer of the mismatch repair protein, MutS
• Mismatches cause distortions in the DNA strand, MutS scans for those
• MutS embraces the mismatch containing DNA, induces a pronounced kink in the DNA
and a conformational change in itself
• MutS has an ATPase activity, but its role in repair is not properly understood
• The MutS-mismatched DNA complex recruits MutL, another protein of the repair
complex
• MutL activates MutH, an enzyme that induces a nick in one strand near the site of the
mismatch
• Nicking is followed by unwinding of the DNA towards the site of mismatch by a specific
DNA helicase UvrD and an exonuclease progressively digests the single displaced strand
• The gap is filled by DNA Pol III and sealed with DNA ligase

14. How does the system know which strand to
repair?
• a wrong decision can lead to permanent setting of the mutation
• The solution in E. coli: Transient Hemimethylation
• Dam methylase methylates A residues on both strands of the sequences 5’-GATC-
3’, which is widely distributed in the E. coli genome, about once every 256bp.
• All these sites methylated
• When the replication fork passes a section with both strands methylated, the
resulting daughter duplexes will be hemimethylated, ie methylated only on the
parental strand.
• Hence, until Dam methylase catches up and methylates the daughter strand, the
new strand is marked by the absence of a methyl group, and hence can be
recognized for repair
• MutH can bind at such hemimethylated sites

16. • MutH binds at unmethylated strand and initially its exonuclease
activity is normally latent
• Only when MutH is contacted by MutL and MutS located at a nearby
mismatch (which is likely to be within a distance of a few hundred
base pairs) does MutH become activated
• How they interact in such long distances is uncertain, but evidence
suggests that the MutL-MutS complex leaves the site of mismatch
and moves along the strand to reach MutH at the site of
hemimethylation
• MutH then nicks the unmethylated strand selectively

17. • Still, removal of single stranded DNA between the nick site and the
mismatch site left to be done
• For this, different exonucleases are utilized depending on the site of
the mismatch with respect to the nick
• If the DNA is cleaved on the 5’ side of the mismatch, exonuclease VII
or RecJ, which degrades DNA from 5’3’ direction is used
• If the nick is on the 3’ side, exonuclease I does the job
• DNA Pol III fills in the missing sequence

19. In Eukaryotes?
• Similar mechanism using homologs of MutS (called MSH, or MutS Homologs) and
MutL (called MLH and PMS)
• Eukaryotes have multiple MutS like proteins with different specificities
• Ex: one specific for simple mismatches while other recognized small indels
resulting from “slippage”
• MutH is absent in eukaryotes as well as hemimethylation and most other bacteria
are unable to utilize hemimethylation and lack Dam methylase as well
• Lagging strands produce Okazaki fragments, and before ligation, the space
between these fragments serve as nicks
• Human homologs of the of the MutS (MSH) interact with the sliding-clamp
component of the replisome and would thereby be recruited to the site of
discontinuous DNA synthesis on the lagging strand.
• Interaction with the sliding clamp could also recruit mismatch repair proteins to
the 3’ (growing) end of the leading strand

20. DNA Damage
Spontaneous Damage by Hydrolysis and Deamination
• Chemical/physical mutagens can cause DNA damage
• But DNA cam also go spontaneous damage from action of water (although aq.
Environment is needed for proper double helix)
• Most frequent and important kind: Deamination of the base cytosine
• Under normal physiological conditions, cytosine can go spontaneous deamination
and thus generating Uracil, which is unnatural in DNA
• C would have paired with a G, but now U pairs with A
• Similarly, deamination converts adenine to hypoxanthine, which bonds with
cytosine, and guanine is converted to xanthine, which pairs with cytosine, albeit
with only 2 H-bonds
• DNA also undergoes depurination by spontaneous hydrolysis of the N-glycosyl
linkage, and this produces an abasic site

21. • All these changes create unnatural
changes in the DNA
• The presence of unnatural base makes it
possible for the repair system to recognize
errors
• Evolutionary significance of Thymine
being present in place of Uracil in DNA: If
Uracil was a natural base, deamination of
cytosine would form another natural base,
impossible for the repair mechanism to
detect and correct

22. What if deamination gave rise to natural
bases?
• If deamination gives rise to natural bases, the repair system would not be
able to detect them
• For example: 5-methylcytosine is really common in vertebrate DNA.
• Its deamination produces Thymine (a natural base), not Uracil
• So, the change is not detected by the repair system
• The Thymine will pair then pair with Adenine while forming a new
daughter strand, leading to permanent fixation of the mutation (called C to
T Transition).
• In fact, methylated Cs are hotspots for spontaneous mutations in
vertebrate DNA

23. DNA is damaged by alkylation, oxidation, and
radiation
Alkylation
• Transfer of methyl or ethyl groups to
reactive sites on the bases and to
phosphates on the DNA backbone
• Alkylating chemicals: nitrosamines
and N-methyl-N1-nitro-N-
nitrosoguanidine, a potent lab
mutagen
• Most vulnerable site for alkylation:
keto group at the C-6 of guanine,
which forms O6-methylguanine,
which mispairs with Thymine,
changing G:C pairs to A:T pairs in
daughter cells

24. Oxidation
• DNA is subject to damage from reactive oxygen species as well (O2
-, H2O2, •OH)
• Generated by ionizing radiation and by chemical agents that generate free
radicals
• Oxidation of G forms 7,8-dihydro-8-oxoguanine, or oxoG, which is highly
mutagenic as it can base pair with A as well as C. If it pairs with A during
replication, it gives rise to a G:C to T:A transversion, one of the most common
mutations in human cancers
• Maybe this is one primary way ionizing radiation and oxidizing agents cause
cancer

25. Radiation
• Radiation of nearly 260nm (UV) strongly absorbed
by the bases, one consequence of which can be
photochemical fusion of two adjacent pyrimidines
on the same chain
• If two thymines fuse, a thymine dimer is formed,
which contains a cyclobutane ring generated by
links between carbon atoms 5 and 6 of adjacent
thymine molecules
• If C is adjacent to T, a T-C adduct is formed linking
C6 of T to C4 of C
• Such dimers are incapable of pairing and stop the
movement of DNA polymerase during replication
• Assays to detect DNA damage: Immunoblotting,
Comet Assay (Single cell electrophoresis), Cell
survival assay.

26. Radiation: X and γ-rays
• Ionizing radiation can introduce double strand breaks in the DNA and
severely damage the chromosome
• Can directly attack the backbone of the DNA or create ROS which, in
turn, react with the deoxyribose subunits
• Cells require intact chromosomes to replicate their DNA, so targeted
ionizing radiation is used to damage the DNA of cancer cells
• Anticancer drugs like Bleomycin cause breaks in DNA. Such agents are
called to be clastogenic (Greek clastos, which means broken)

27. Mutations Caused by Base Analogs and
Intercalating Agents
• Base analogs: structurally similar to
proper bases but differ in ways that make
them treacherous to cell, thus can get
taken up by cells, converted to NTPs, and
incorporated into the DNA during
replication, but base-pairing occurs
inaccurately, leading to frequent mistakes
• 5-bromouracil: one of the most mutagenic
analog of Thymine
• It mispairs with Guanine via the enol
tautomer (In case of Thymine, the keto
tautomer is more favored, but for 5-
bromouracil, the enol tautomer is more
favored)

28. • Intercalating agents: Flat molecules with
several polycyclic rings that bind to the
equally flat purine or pyrimidine bases
• They may cause insertions or deletions in
strands
• Insertion: The DNA polymerase reads the
inserted area as more than one base pair,
hence, inserts more bases opposite to it
than actually needed
• Deletion: DNA polymerase skips the
intercalated region and in the process, the
base that the agent had bound to

29. Repair and Tolerance of DNA Damage
• DNA damage can have two types of consequences:
1. Thymine dimers (irradiation), nicks, breaks in the DNA backbone
impede replication or transcription
2. Other kinds of damage (base analogs, intercalation, alkylation,
oxidation, deamination) can cause permanent alterations in the
DNA sequence of the progeny cells
• Cells have evolved elaborate mechanisms to identify and repair DNA
damage before it blocks replication or causes a mutation

30. Direct Reversal of DNA Damage
1. Photoreactivation: DNA photolyase catches energy from light and
uses it to beak down the covalent bonds linking adjacent
pyrimidines (dimers)

31. Direct Reversal of DNA Damage
2. Removal of methyl group from methylated base O6-methylguanine
that pairs with T (resulting from alkylation) by the enzyme
methyltransferase which transfers the methyl group to one of its
cysteine residues (after that, the enzyme is not used again)

32. Base-Excision Repair and Base Flipping
Mechanism
• The most common way of “cleansing” DNA is to remove and replace the
altered base
• Two principal ways: Base Excision Repair and Nucleotide Excision Repair
• In Base Excision Repair, an enzyme called glycosylase recognizes and
removes the damaged base by hydrolysing the glycosidic bond.
• The resulting abasic sugar is removed from the DNA backbone in a further
endonucleolytic step. Endonucleolytic cleavage also removes apurinic and
apyrimidinic sugars that arise by spontaneous hydrolysis
• a repair DNA polymerase and DNA ligase restore an intact strand using the
undamaged strand as a template

34. There are more than 1 type of DNA
Glycosylase
• Depending upon the damaged base to be repaired, there are different
kinds of DNA Glycosylases, 11 have been identified in human cells
• A specific glycosylase recognizes uracil (generated because of
deamination of cytosine), and another is responsible for removing
oxoG (generated because of oxidation of guanine)

35. How do DNA Glycosylases Detect Damage?
• Each base is buried deep in the DNA helix
• Evidence shows that the enzyme diffuses laterally along the minor groove of the
DNA until a specific kind of lesion is detected
• DNA molecule shows great flexibility, the damaged base is flipped out away from
the helix where it sits in the specificity pocket of the glycosylase as seen in X-ray
crystallography
• Base flipping is done without much structural changes to the DNA structure, and
hence, the energetic costs may not be all that much
• Nevertheless, it is unlikely that glycosylases flip out every base to check for
abnormalities as they diffuse along DNA. Thus, the mechanism by which these
enzymes scan for damages is still unknown

37. What if the damaged base is not removed
before replication?
• Fail-safe glycosylase
• Ex: in oxoG:A pairing, if the oxoG base is not removed before
replication, a fail-safe glycosylase removes the A that it pairs with in
the daughter strand and replaces it with C
• Similarly, 5-methylcytosine gives Thymine upon deamination, which
can incorrectly pair with G
• The glycosylase system assumes, so to speak, that the T in a T:G
mismatch arose from deamination of 5-methylcytosine and
selectively removes the T so that it can be replaced with a C.

39. Nucleoside Excision Repair
• This system works by recognizing distortions to the shape of the
double helix, such as those caused by a thymine dimer or by the
presence of a bulky chemical adduct on a base, rather than by
recognizing a specific base
• A chain of events removes a short ssDNA patch that includes the
lesion, and the gap is filled by DNA polymerase
• Accomplished by 4 protein UvrA, UvrB, UvrC, and UvrD, types of
exonucleases

40. • A complex of two UvrA and UvrB molecules scans the DNA, the UvrA subunits
detect the distortions
• After detection, UvrA subunits leave the complex, the remaining UvrB complex
melts the DNA and creates a ss bubble around the lesion
• The UvrB dimer recruits UvrC, UvrC creates two incisions, one located 4 or 5
nucleotes 3’ of the lesion, and the other 8 nucleotides 5’ to the lesion
• The 12-13 residue long lesion containing DNA is removed from the rest of the
DNA by a helicase UvrD
• Uvr: because this system repairs damage from UV light, mutants of the uvr gene
are sensitive to UV light and lack the ability to remove T-T dimer or T-C adducts,
and bulky lesions of many kinds
• In humans, xeroderma pigmentosum genetic disease results from a mutation in
uvr genes or inability to repair UV induced damage, which renders them very
sensitive to sunlight, making them prone to skins lesions, and even cancer

42. • In higher cells, the principle behind nucleotide excision repair is largely the same
as in E. coli
• But the machinery involved is a little more complicated
• Repair and excision involves 25 or more polypeptides (enzymes)
• XPC: equivalent to UvrA (detecting distortions in the DNA)
• XPA and XPD: equivalent to UvrB (forming bubble around the lesion) with the ssb
protein RPA
• The bubble creates a cleavage site for a nuclease called ERCC-1 XPF
5’ to the lesion and 3’ for the nuclease XPG (eq. to UvrC)
• The excised strand is 24-32 nucleotides long, and as in bacteria, gap filling is done
by polymerase and ligase

43. Transcription Coupled Repair
• Nucleotide Excision Repair also rescues RNA polymerase, the progress
of which has been halted due to a distortion in the template strand
• NER proteins recruit to the stalled RNA polymerase during
transcription
• In this effect, RNA polymerase acts as a damage sensing protein
• The TFIIH protein that unwinds the DNA during transcription, includes
two subunits, which are in fact XPA and XPD
• So, helicases of TFIIH melt the DNA around a lesion during NER and
also during transcription

45. Double-strand break (DSB) repair pathways
• How do cells repair double-strand breaks in DNA in which both strands of the
duplex are broken?
• DSB repair pathways accomplish this
• One recombination based pathway retrieves sequence information from the
sister chromosome. Recombination based DSB pathway (another chapter in the
book)
• DNA recombination also helps repair errors in DNA replication (by retrieving seq
information from another daughter molecule of the replication fork and
completing the recombination after which the NER does its job
• or by producing a break while passing over a nick, which can be then repaired by
DSB repair pathways
• Maybe recombination evolved to repair DNA damage as its primary function

46. DSBs in DNA Are Also Repaired by Direct
Joining of Broken Ends
• A DSB is the most cytotoxic of all kinds of DNA damage
• Consequences if left unchecked: blocking replication, chromosome loss, ultimately death
of the cell or neoplastic transformation
• DNA damage repair pathways are overlapping, so recombination alone is not responsible
for mending DSBs
• Recombination relies on sister chromatid’s template for repairing, but what if a non-
replicated chromosome suffers a break?
• An alternative DSB repair system comes into play: Non-Homologous End Joining
• NHEJ is a backup system in yeast, which primarily relies on recombination bases DSB
repair, but in higher cells, it is the primary way
• NHEJ protects and processes the broken ends and joins them together, but broken ends
lose sequence info. Thus NHEJ is mutagenic, but the effects are far less hazardous to the
cell than there are consequences to leaving broken ends unrepaired

47. How NHEJ works
• It doesn’t involve extensive stretches of homologous sequences
• Two ends of the broken DNA are joined by misalignment between single strands
protruding from the broken ends
• Misalignment because of pairing between tiny stretches of complementary bases,
nucleases remove the tails and polymerase fills the gaps
• Till date, seven proteins of the NHEJ pathway have been identified in mammalian
cells: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, Cernunnos-XLF, and DNA ligase IV
• Ku70 and Ku80 are the most fundamental components: constitute a heterodimer
that binds to the DNA ends and recruits DNA-PKc (protein kinase)
• DNA-PKcs, in turn, forms a complex with Artemis (a 5’-3’ exonuclease that is
activated by phosphorylation by DNA-PKcs
• These nucleolytic activities process the broken ends and prepare them for
ligation.
• Ligase IV performs ligation in a complex with XRCC4 and Cernunnos-XLF

49. NHEJ in bacteria
• NHEJ less frequently observed in bacteria
• In Bacillus subtilis, a Ku-like protein and a DNA ligase is produced when it sporulates and it
packages the protein into a mature spore
• This two protein NHEJ system repairs heat-induced DNA breaks when the spore germinates
• Mutants lacking these spores barely survive harsh conditions
• Spores have only one chromosome, so it makes sense that DNA breaks are mended by
NHEJ, not recombination based methods
• The doughnut like structure of the spore chromosome makes the broken ends lie close to
each other, facilitating joining
• NHEJ is the reason why B. subtilis spores are able to survive harsh environments

50. Tolerance of DNA Damage: Translesion
Synthesis
• DNA repair systems are not foolproof and damaged bases can be
encountered by a DNA Pol during replication
• If replication ceased, it can be more damaging
• A fail-safe mechanism to bypass these damages and “tolerate” them
• One mechanism: Translesion Synthesis
• A highly error prone mechanism but spares the cell a much worse fate
of incompletely replicated chromosome
• Tolerance leaves the lesion intact, which can be corrected later by
other pathways

52. Translesion Synthesis
• Catalyzed by a special class of DNA polymerases that synthesize DNA
directly across the site of damage
• In E. coli, DNA Pol IV (DinB) or DNA Pol V (complex of proteins UmuC
and UmuD’) perform the job
• DinB and UmuC are part of a distinct family of DNA polymerases
called Y family
• In humans, out of 5 known translesion polymerases, 4 belong to the Y
family

54. • Translesion polymerases incorporate nucleotides independent of base
pairing, that is why synthesis can occur over a damaged strand
• As they do not read from the template, the process is highly error-prone
• Even for apurinic or apyrimidinic sites in the template, nucleotides are
incorporated
• However, not completely random
• DNA Pol η correctly inserts two A residues opposite a thymine dimer
• Structural studies show that the active site of DNA Pol η is more
accommodating of thymine dimers than DNA Pol κ, another translesion
polymerase

57. • Because of high error rate, transleion synthesis and NHEJ are systems of last
resort
• The price paid for allowing a cell to survive and reproduce: high levels of
mutagenesis (fixed in the genome)
• So, synthesis of translesion polymerases an enzymes are highly regulated
• Ex: in E. coli, these polymerases are synthesized only in response to DNA damage
• SOS Response: DNA Damage leads to the proteolytic destruction of a
transcriptional repressor (the LexA repressor) that controls expression of genes
involved in the SOS response, including those for DinB, UmuC, and UmuD
(precursor of UmuD’). The same pathway is also responsible for the proteolytic
conversion of UmuD to UmuD’
• Cleavage of both LexA and UmuD is stimulated by a protein called RecA, which is
activated by single-stranded DNA resulting from DNA damage. RecA is a dual-
function protein that is also involved in DNA recombination

58. How a translesion polymerase gains access to the
stalled replication machinery at the site of DNA damage
• In mammalian cells, entry into the translesion synthesis pathway is triggered by
chemical modification of the sliding clamp
• Ubiquitination of the sliding clamp: this makes the sliding clamp recruit a translesion
polymerase (it contains domains that recognize and bind to ubiquitin)
• The translesion polymerase then displaces the replicative polymerase from the 3’ end
of the growing strand and extends it across the site of the damage
• Ubiquitination is a distress signal which helps rescue a stalled replication machine
• In addition to this polymerase switching mechanism, a synthesis that used gap filling
mechanism has also been reported
• The replicative DNA polymerase skips over the damage introducing a gap, which is
filled by the translesion polymerase

60. Unexplained questions:
• How exactly does the translesion
enzyme replace the normal replicative
polymerase in the DNA replication
complex?
• How does the normal replicative
polymerase switch back to and replace
the translesion enzyme at the replication
fork? (maybe the translesion enzyme has
low processivity)

61. THANK YOU.