Learning outcomes

DNA
DNA Damage, Repair and Mutation Exogenous sources ~ Endogenous -cellular processes
.. I
~: p : ii:feren)t ty~:.:.: DNA damage

mechanism \
Damaged DNA that
escape repair
I mechanisms
Prof. W.S. S Wijesundera
Term ID t= DNA replication :.:.::--~
AL/2013 Batch Normal DNA DNA mutations~
August 2015 Different types of mutations 2

How does DNA damage occur? Endogenous DNA damage

/~
Results of natural
cellular rocesses
•Base damage
Modification of bases- eg. Deamination
Tautomerizat fon
Induced
Loss of bases- eg. Depurination
(Environmental)
l •Replication errors : Polymerase misincorporation

•Recombination errors : Unequal cross over etc.

•Byproducts of metabolism : Oxygen radicals

Sohlatic cells include superoxide (02t' ). hydrogen peroxide (H202),

+ +
Affects
hydroxyl radicals (OH ' ) and singlet oxygen (0)

Inherited by •Alkylation : eg . reaction of S- adenosyl methionine with

offspring (all cells) individual only DNA

Deamination

u
't'.
=~-::
.);
cNI
~
c,....,.,e;
- ;6 ~

',.lt~ jiJ \
dMtf,r..-W,

= ~= ··- + = ~=
I~

- -+
-
.~
a -··-

_ H_
~~11():l ~Otl
- c-
=;= -- =;= -+
Deaminated cytosine pairs with ... A,, ....? -·-
Can cause Base conversions

_j
 De urination
Tautomerization f' -. . . . 1
!
I

Tautomerization is rare
-NH2 AMINO ... imino =NH
=O KETO ... enol -OH

...~ r
+:J-?"rtSQ
ICetoFonn Enol f'orm
N- Glycosid,c
bond
Amino Form lmll\O form SQonta~eous reecrioo
•• • ;Jp IO 10.000 Pl.me bases per cell in e day
. ~-,.s ~., cell ,n a oay
~ Up 10 500 Pynr.11drte - ~ llC1WOfl ,r nO! repaired
.)- Cr!oelM
.,.,..,,. Al>tSIC sites are nOf\-eO<Mg and l>k>Ck rep

.
..... 'if

"
During replication , DNA pol~merase will incorporate a rand~m
C (imino) C:A
base opposite the apurinrc srte
G (enol) G:T

1<ep11cat1on errors Induced DNA damage ,

- ---~ - . ..,_ -- --- --· --- ·- ------ -·- --- -- -·-- ---
·- · - - ·- --- ...
Exogenous DNA damage
DNA polymerase is not perfect !
• Chemicals •Natural - in food - aflatoxin ~
~. ~{·
~
~:·:
.
·-.
-
•-.
'
(mutagens) . .... -~: -..."'
Replication errors are the main source of mutations J •Food preservative - nitrites ·' · .-
by digestion of food containing preserwt1ves
...--.. .·
base pair Insertions & •Benzopyrene- smoke from cool, autos, cigars
mismatches deletions
•Alkylatng agents -alkylate Nor O atoms in the
c,- 04,,, ..~,
bases-
Due to }~ ~Ck.
Eg. Methyl bromide, ethylene oxide,
1/1,000,000 wrong
base added
Strand slippage ...............
O · -CH,· ··Ot,
Nitrogen mustard -WWI -nerve gos
during replication •Intercalating agents-
Repeat regions more pron, EtBr used as a dye to stain DNA
•Agents that alter DNA structure -
9 Large molecules which bind to bases in t>NA
10

Exogenous DNA damage cont ..... Food preservatives

Radiation
Nitrous acid Produced by
•Background radiation : varies with geographic location
(HN02) ~ digestion of
From natural sources: include
• cosmic rays from the sun and outer space
• radioactive elements in soil wood and stone
! Nitrites
•In the atmosphere (radon)
Deaminating agent
•Artificial sources of radiation: Humans have created:
contribute to our radiation exposure ....
Adenine _,. Hypoxanthine
• Nuclear testing and power plants
•Medical testing (diagnostic X-rays and other Guanine - Xanthine
procedures)
Cytosine _,. Uracil
•Various other products {TV' s, smoke detectors,
airport X-rays)
CH 3Cytosine ---+ Thymine
11
12
 7,
- F

Intercalating agents ~J. .

•
Benzopyrene
Planar, aromatic molecules ,"Y~ Polycyclic aromatic hydrocarbon
.,,l..._,,,l-.✓<~·~
Inserted between base pairs
causing "stretching" of the
DNA duplex
6 forms
DNA ADD CTS

Examples: 'dil\e orange,

proflavin, ethidium bromide Bulky lesions

Insert on extra base during

ONA replication Nucleotide substitutions /deletions
Chromosomal rearrangements etc ••
"

_________ A lkylati ng_agents __ ________ _ Radiation

Forms the LARGEST group of mutagens
•UV radiatio< \
Cannot penetrate beyond ,the outer
layer of the skin
•Ionizing radiation

•
alpha(a)particles
Highly reactive •
beta(~) particles
.... Guanine-O 6 methyl guanine
• gamma ('y) parttcles
• xrays

-·- l
Base pairs with T
15
Penetrate the whole body can cause
somatic and germ-line mutations 16

~.•
6 --
Ionizing radiation Radiation from x-ray
machines
Direct damage -D A strand cleava e: one or both strands
can lead to rearrangements, deletions,
chromosome loss) •Computed Axial Tomography
-damage to/loss of bases (CAT Scan. CT Scan)
-cross linking of DNA to itself or proteins
• MGrnmogrophy
Indirect damage- radJ --~- eel
Water dissociates
under Alpha "There is no safe threshold"
radiation to
hydrogen radical
and "Need to prove that low doses of radiation
a hydroxyl radical are not harmful."
,.
0 t7 ,.
 d.- mer _ I+ p fe,., t hcm c.1
In Ci .J> Nf"t •..,,,o f ~ c u l ~ /

Qt1 d 0 Jr-,l"\ ,r'\ e

Types of DNA damage

Ultra violet radiation •.-1~

·\ = -- ---:r1
_....,., -(!)~ "'-~9 Chc111UI crou-l!fllu
i "'b,., ..••' ~r•n~
- )'.~);{
~
OM
·,. '\ \ • <-+-~
· • T/"
~
,..· i--~· ---4- ... .,_fl...
f ·,.. 5 ' --CCGAAttCAG--3 ' •· r• • H\'d"'"'"" ~
3' --OOC'l''l'AAO'l'C--5 ' lY)'ri!hldfoic ,11,
,.... -·· ." .

Pyrimidine dim£rs ,3_ ,. , I

· !'\- ~ I

Deoxyr1bose sugar •• · , ~
O Phosphate /
• R- alkyl 9'""1' ,L . , 20
19

Frequency of DNA damage

*"'·
Mtd,yl6........
. .,000 JU You are NOT
* Ofpurloatioll l4,000 JO..! expected to
memorize these
*O'· J,UO u figures.
Meti,ylGu"'!ino
* OaldlndOHA 2,ao 1.Z
* Dtpy,tn,idation J.J20 0.5
•Activation of a cell cycle checkpoint
*'~ 3'0 O..!

.... •Commencement of transcriptional programs

dNffllnatlon
,rr D••l1 ....... o;lll • Carrying out DNA repair
' •Initiation of apoptosis when the damage is severe

DNA Repair Damage reversal

R'educes potential mutations fo accepta&re fevers
• Several re~ir systems Photoreactivation
/ "-..
Ligation of single strand
• Specific for. TYP of damage A single enzyme breaks
(photolyose enzyme)
•Damage reversal • Photoreoctivation (Not in IIICllllmals)
breaks the covalent
Simple breaks in one strand
ore rapidly repaired by DNA
• Ligation of single strand breaks bond of pyrimidine ligose
dimer in presence of
•Damage removal • Bose excision repair light. Dmages caused by X-rays and
• Nucleotide excision repair some chemicals like peroxides
• Mismatch repair Not found in Human
•DNA damage
tolerance
, Recombinational repair Will
,Mutagenic repair NOT
I Blootn sylldromc:inwlved with
DNA ligose deficiency
be dtSCUSSf!d
23
 C>omoge r emoval I Ex ci sion r epair
•Bas• •xclslon repair Bas ic s teps.Recog n, zc damage
-Remove damage by e >(cis,ng part of - st rand
The d411'ogcd or inappropriotc bo:IC 1, rcr11ovcd frOffl -DNA pol. f ill gap -usi ng the complementary strand
its wgar llnlcagc ond replaced -Ligate to restore contil'IUity of DNA

•Nucleotide excision repair iBose ex cision repa ir Nucleotide exc,s1on reporr

Repair ()NA do111ogc which is "bulky" . Several nucleotides
Repairs - R~irs -
,_,r the domoged DNA ore olso remo~ lffllf I epleu.d .._ pyrimidine d,mcrs
Methylated
dcominatcd chemical odducts-
abosic sites benzopyrinc, aflato x,ns,
•Mismatch repair chemotherapeutic agent s -
oxidised bases
Occurs t r t>N~ replication os a last "spell-check" cisplatin
on its accuracy .

l Base excision repair Uracil N Glycosylase

(Specific example for base excision repair)
[l Damaged I abnormal base
•Uraci l in DNA comes mainly from deamination of

l~ AP endonuclease
1

.....

cleaves backbone n
removed

I /
(Leaves AP site) /
{'-p,tr -r
..J
C

~
~
1, ~ • '" • ·f
...... ,;;.~~1.cQ.
•Uracil N Glycosylase enzyme removes uracil from
DNA
•Resulti ng abasic s ite (AP site)
(lea~fis nucleotide gapv CJ-e,f Fill in the blanks

t:a Fs.:J
t:8 ~ DNA Pol synthesize new DNA
DNA ligase seals nick
If uracil is not removed it will pair with ...fr. ...
causing .... ... to ...T..... transition in subsequent replications
C

27 211

Nucleotide exc1s1on repair Nucleotide excision repair

Repairs large lesions (structural changes to helix)
Eg: T-T dimers Eg: T- T dimers
Benzopyrene -adducts 5'--------CCGAAttCAG-------3 '
3'--------GGCHAAGTC-------5 '

•
Damaged DNA recognition
I
ilNCISlON
-
~
)(
::J a,
Incision followed by excision removes
S' ------~ ✓CCGAAttCAG' '------3 '
1 1

E- 3 ' --------GGCTTAAGTC-------5 '

>- .a. o/igonucleotide (27- 29 nucleotides in the
..o E ,I. EXCISION
0
+- u damaged area in humat
::JC
0 ·-
-0 a, DNA Pol. synthesize new DNA
s•-------- -------3 '
3'--------GGCTTAAGTC-------5 '
a, +-
·-L L
0 (genetic information from the other strand)
a .a.
u
~&AP -FllLlNS (BY A POI.YMERASE)
5 '- -------CCGAATTCAG-------3 '
DNA Ligase ! r s nicks 3 '- -------GGCTTAAGTC-------5 '
'.!O
 ~-

Mismatch repair
•S cialized type of nucleotide exc;ision repair -
re":oves replication _errors
•Mismatches causes distortion of the double e I!
helical structure (there is no damage or modified base Generally, in the geno~ ted at GATC sequences
i
but the wrong base) Ne of adenine is methY a
MJIINllrecl.,... in ...,.,,
r;- ~t - , , ,, i , , ,,
I..~ - :-- -·---~ ft'
!!Yftt.,._oed ONA alrand
'
f
I
___.,,...T, T J i • ' ~
f j. f
..,_...
I
_
f
01'f ~
• f
. .. - ~ . , . . . _ . . . .
•
,,.,_
;r
a.:
"'
- .L.,. ....,,,___,.______ ., . _ - C T ~ O ·Methylated
3
• Themajor difference between mismatch repair and
damaged DNA repair- L--~G~----L------..-;,;.:. ._ _.1, adenine
in parent
strand
recognize which base to excise · h · ethylated DNA
' I o✓ " Immediately after replication emim
( Ol"I<!- r'Yt ~ parent: .s-f"(lin"-.
au5~i'1r stm J - - - - - - -

fare-tt t--sttanck ~ I Gt' h,, mG-lti.jlc1+ecl

1 Mut proteins identify and bind Repair of double strand breaks

mismatch and methylated parent

"•;. ;, . 1
. .........,-
~• H
..XMf"w
strand on
hemi-methylated Double strand breaks commonly caused by
DNA igh .energy raoiation
-··
z.. .. .
~ .
a !l~I'i

. , -~ .•. fl

,,,.,. ...,<
-l
<''r°Aa ·
-·~·
..,.,
..._. .._.._._,.,
., .
-'

..,,..
. .. .... _ T
.
•.
~

...........
2
Exonuclease cleaves
daughter strand in
region of mismatch
3 DNA polytnerQse
Gxidati.o ree radicals

TWO key pathways for repair

7ii"_._.__._:·- ___-.,~
([';..:' .c,,,,. filts the gap and
~ ,,,, DNA ligase seals ~coJrtt>i.h((tioh. cepair-
(er.rQr-free]
- - -~ ; ~
C
'!""""" ... '!"- -

t<emoves m1sma1cnea bases in DNA with 99.9%

..
... ~, • • ..
- f -

33
efficiency-' carried out by multi protein complex

DNA Damage , Repair and Mutations

™' .,-., ., ;.;:. .11.End-joining
....
repair
:.,..,.n
DNA--
t'~...... Endogenous •cellular processes
f" - Exogenous sources

• jo..... ........
L :=··.:
ji Nucl~es remove a few
bases from the ends
...... DifferenJt
...,.Repair
ty~~ of DNA damage

\.
""...t(O,m] ._ : •·-]
...,ll'"'0111 . mechanisms \
t>amaged DNA that
Escape repair

~ ·
•/•-_[" t 3E~sar~joined t~~th~r-by -,
- -- . hgase j I ·· mechanisms
,.,111'1'io,.,.o....o-1-n-n-u-n-n --·- -· ·
Few bases always lost ct site of repair Normal DNA DNA mutations
Occasionally 'wrong ends joined- chromosomal translocations
Different types of mutations *
 A'l lllliiillill________
___.

MUTATION Mutations
Stable (heritable)
change in the nucleotide
sequence of the genetic
material
•Involve less than 1000 bp

•Often it is a single bp chang

Good effects? •Observed cytogenetically (point mutation)
(at the level of Chromosomes) - substitutions
deletions
•Include - deletions, insertions
Bad effects ? duplications
inversions,
translocations
,.

Molecular nature of point mutations Triplet expansion

Normal (wild type) Increase in the number of repeating triplets
--&MTTC- ·
;_ --CTTAA&·· - - - - -6.AGTTC- Repeat Normal Mutated
Substitution: / ·-CTCAAG- repeat number
sequence
-6ATTTC-- A-G Fragile..X syndrome CGG 6-29 >500
-crAAAG--
• Transit ions:
A-+ T Purine - Purine
• Transversions: Pyrimidine _ Pyrimidine Huntington disease CAG 9-35 37-100
Purine !::; Pyrimidine
Huntington disease- neurodegenerative disorder-

IDeletion: I :~if=-:
'+ ... I -GAACTTC-
Insertion:
accumulation of protein aggregates

Same amino acid is encoded in the polypeptide (poly glutamine

-CTTGAAG-
"°

Nucleotide substitutions can give rise to SILENT MUTATION

different types of mutations
No effect on gene function
Nucleotide change causes No amino acid change
Silent - TGT(Cys) - TGC (Cys)
GCA (Ala) -+ GCN (Ala) No phenotypic change
Missense TGT (Cys) - TGG (Trp)
Most likely happen in the 3 rd position (wobble base)
Nonsense TGT (Cys) - TGA (Stop) of degenerate codons

.,
 MISSENSE MUTATION NONSENSE MUTATION
Base pair substitution- Nt change results in
Mostly base poir substitution- Nt change results in
'STOP' codon
amino acid change
p- globin gene
Truncated protein
1
DNA: CTC CAC
_ _ __, ATC
RNA: GAG GUG DNA: ATG
AA : Glutamate > Valine RNA: UAC ----,>~ UAG
Tyrosine --~> STOP

o:
AA :
er•"-•
.J • • Nonsense mutation changes the amino acid encoded codon
to a stop codon- premature termination of translation of the pr';1ein
Sickle cell anaemia

What is a reading frame? FRAMESHIFT MUTATION

The sequence of nucleotides In mRNA can be read in three
Insertion I Deletion of single nucleotide results
READING FRAMES
in change of reading frame
t
Out of frame translation of all of the
down stream codons- different amino acid sequence
t , , I I I • J , I I I I , IS , J , 1 I I I I . : :
GUCAllGUUH,\OCc;CA,\UCAOOAAO UOU

!
.&of' ::r ~ t..-,,, .;

6l1J~b~JAG66b~~t,bl/;oA~il ,~~'
1'!1- <l' • O ~ r 0:l y st,
••• ••• ••• ~ m
··~ • • • • • • 11
Identify the reading frame of the following eukaryotic mRNA
•• II
5' GUACCUGAUMUGACGUCGGUACAGAUUACGUUGAUACAGCAUGAAA

AUGACGUCGGUACAGAUUACGUUGAUACAGCAUGAA

Frameshift example Point mutations causing p Thalassaemia

Promoter tu _ _ _____ ______,
NHZ- MET- Thr- Leu- Lys- COOH 5' & 3'UTR r· ··· f ... . ........ ... ..... .,. .... .. .. .... :··ni
RNA procesaing~:::;::;:=:;::====::::;==:
5' -AUG ACC UUG AAA UAA 3' ATG .
Nonsense ~;::=:::::::========::::::;:::::::
, ,, ,
J Deletion of A ' Stop codon Frameshift•(:7C:" iWI

NHZ- MET- Pro- COOH Gene deletions causing a Thalassaemia

5' -AlN CCU UGA AAU AA 3' -- ~ ,a-~ '.i: J ;·1: ·

r :• : l
•,..t.._, _
~l · ""
✓At· ·
t -!l• !
•4
'. • ~ 1 •
.Y.x;~- r
.. ~ ; ..<,,-~
, /J. •

Stop codon .. ~-.v. j '} ;t.t·,

- .·~ . . , .

. . A•~-; • ,. {:.#1
47 ~.,-t.-,.r,• s ~1 • ~r•~ ) 11-1¢.- •d t r,."9 ; ~ .-..1~ ~
J -~.JCtt,.~;r,;'tli ·~- '.'\, ; - .. "\.f(,✓ ~""fl''!• :-~t "t')(:t:t.. '< t
 1/
I

Defects in r ~p~ir pathw~)'S lead to <:=_oncer

DNA repair diseases
Mutations accumulated in several genes ,...cc
J
Somatic cells
· HcridltGI')' nonpolyposls col-ckGI ccnur
· Autosomol d-illClnt, inultigenic, up to 1/Z00
. 5y,llpto111• !
Germline ~ e l l division • Hi9h fr,q-,cy of colon ond -..-ol other cone,..

~ Cause by
Eg: Breast cancer
"Loss of function" mutations in genes
Inherited defects in BRCA 1, BRCA2 genes involved in mis11Jcrtch repair pathway J
(recombination repair pathway) 50
predisposes women to breast cancer

UV radiation

fncreasedtutations

f Frequency of
sunlight induced
skin cancer 51
 DNA DAMAGE, REPAIR AND MUTATION

• Mutation - Stable(heritable) change in the nucleotide sequence of the genetic material

• Good effects and bad effects
• If mutation occurs in a germ cell, it is inherited by the offspring but if it occurs in a somatic cell it affects that ind_ividual only

DNA DAMAGE

Spontaneous Induced (environmental/exogenous)

Spontaneous Replication Recombination Chemicals Radiation

base damage errors errors
~

~ ~ l
Deaminating agents UV-Thymine
Ionizing radiation
Intercalating agents
dimers
Alkylating agents a /3 V x -rays

Loss of bases Modification Most common Unequal Agents that alter DNA structure
of bases source of mutations crossovers
~ *DNA polymerase

l
Depurination
~

Deamination
C --.- U
~ (Base pair mismatches, insertions and deletions)

Tautomerization
(rare)

!
Depurination >>
amino < >
imino
Depyrimidination
keto < )'
enol
C (imino) C: A
~
Removal of base
G (enol) G: T
Due to N- glycosidic
cleavage • leaves AP site

© Repeat campaign 2010 A/ L

 - I '

Deamlnatlng agents
Food preservatives
Nitrites - - - - - - - - HN02; nitrous
Digestion
A -+ Hypoxanthine Abnorm a
acid - deaminating G -+ Xanthine
base
agent C -+ Uracil
paring
CH3C • Thymine

Benzopyrene

Smoke from
c?al, auto,r\
Polycyclic
aromatic
Forms
r\
DNA
q Bulky
lesions q DNA repair/
replication r\
Causes nucleotide
substitutions,
cigars L/ hydrocarboh-,' adducts L/ deletions,
chromosome
rearrangements
Alkylating agents
Alkylate (adds methyl or ethyl groups) Nor O atoms in the bases, phosphate of DNA backbone
E.g. Dimethyl sulphate
Methyl bromide
Ethylene oxide
N mustard - WWl -nerve gas

Intercalating Agents
Ethidium bromide
Planar, r\ Intercalating agent DNA replication r\ Causes deletion or addition
aromatic L/ (Inserted between y of bases (Frameshift
molecule base pairs) mutations)

Ionizing radiation
t •
Direct damage Indirect damage

~---~
DNA strand
----·--..
Damage to/ cro"ss linking
+
Radiolysis of the water in the cell
breaks loss of bases of DNA strands +
Free r.oeiatien ~1c e 'i

+
React with DNA & RNA

+
Cell death
 DNA MUTATIONS
Gross_._--------------------.
Fine scale
Observed cytogenetically
Less than 1000 bp
[At the level of chromosome]
Include -- Deletions
Duplications
Substitutions Deletions Insertions [e .g. duplication]
Inversions
Translations
Tra nsition Transversion
~I Leads to frame shift mutations
Purine--. Purine Purine ,. "' Pyrimidine
Pyrimidine - - . Pyrimidine

Missense Nonsense

+
No AA change +
AA change
+
Change results in "stop" codon
No phenotypic change
Most likely 3 rd position
E.g.-
Sickle cell anaemia
+
Truncated protein
(Wobble base) f3 globulin gene
in degenerate codons CTC __.CAC
Glutamate Valine

DNA REPAIR

Nucleotide Excision Repair Base Excision Repair

-Methylated
Recognize damage -Deaminated
(Repairs large lesions) -Abasic sites/oxidized bases

•
Nuclease cleave on both sides of the +
Damaged base removed by DNA glcosy\ase

•
lesion and damaged portion removed
i
DNA polymerase I fills gap using complimentary strand Leavel AP site

+
DNA Ligase seals the nick AP endonucleases cleaves the backbone

E.g.
+
Pyrimidine dimer Leaves nucleotide gap
Chemical adducts
Benzopyrene
+
Aflotoxins DNA polymerase I synthesize new DNA
Chemotherapeutic agents + Cisplatin +
DNA ligase seals nick

© Repeat campaign 2010 A/L

 4

Mismatch repair
• Specialized type of nucleotiae excision repair removes replication errors
• Mismatches causes distortion of the double helical structure
• Major difference between mismatch repair and Damaged DNA repair - recognize which base
to excise
• Repair can distinguish between old & new strand (old strand adenine nucleotides
methylated)
• Removes mismatched bases in the DNA (99.9% efficiency}
• Multi protein complex
• Repair system follows replication fork
• Find "spell check" for DNA replication
• Defects in mismatch repair~ leads to hereditary non-polyposis colorectal cancer (HNPCC),
also called ynch synarome

Repair of double stranded breaks

End Joining Repair Recombination Repair

(error-prone/mutagenic) (error-free)

Repair proteins bind to ends of ds break

+
Nucleases remove a few bases from the ends

~
Ends are joined together by ligase
Few bases are j.1 a s lost at the site of the repair
and occasionally wrong ends are joined-chromosomal translocations

Double stranded breaks are commonly caused by high energy radiat ion and oxidation free radicals

• Why do you think DNA has thymine instead of Uracil?

Uracil in DNA ~ form deamination of cytosine
So has to be repaired- base excision repair

Xeroderma Pigmentosum
Defects in one of several genes involved in nucleotide excision repair pathway (XP genes)
UV radiation (sunlight) -+ -+
1' mutations TT dimmers (skin cancers)

© Repeat campa ign 2010 A/L

I