DNA Mutation and Repair

Endah Wulandari
Biokimia Farmasi (SMT Genap) 2014

DNA Damage
Mechanisms for maintaining genetic stability associated
with DNA replication in E. Coli
Machanism

Cumulative error frequency

Base pairing

~10-1 - 10-2

DNA polymerase actions (including base

~10-5 - 10-6

selection, 3'->5' proofreading)

Accessory proteins (e.g. SSBP)

~10-7

Post-replication mismatch correction

~10-10

Spontaneous alterations:

(a) Mismatches: Occurs

during DNA synthesis (i.e.
replication, repair, or
recombination)

(b) Tautomeric shifts

Nucleotides spontaneously under go a transient
rearrangement of bonding, e.g. a shift from
NH2 (amino form) to NH (imino form) or C=O
(keto) to C-OH (enol). Therefore, if any base
in a template strand exists in its rare tautomeric
form during DNA replication, misincorporation
in the daughter strand can result.

Base Pairing of Imino A-C

(c) Deamination
Three of the four bases normally present in
DNA (cytosine, adenine, and guanine)
contain amino group (NH2). The loss of
the amino group (deamination) can occur
spontaneously and result in the conversion
of the affected bases to uracil,
hypoxanthine, and xanthine, respectively.

(d) Loss of bases

Depurination and
depyrimidination: The loss of
purines or pyrimidines from DNA
usually occurs at acidic pH;
however, it can also happen in
physiological pH (~10,000 purine
per day in mammalian cell; ~500
pyrimidine/day). This will results
in breaking the 3' phosphodiester
bond called -elimination.

Induced Mutations
(a) Physical agents that damage DNA:
--- Ionizing radiation: OH, O2-, H2O2,
damage base and sugar residues.
--- UV radiation: Cyclobutane
pyrimidine dimers, Thymidine dimers
(T-T) dimer

Chemical Agents
(b) Chemical agents that damage DNA:
--- Alkylating agents: Alkylating agents are
electrophilic compounds with affinity for
nucleophilic centers in organic macromolecules.
These include a wide variety of chemicals, many of
which are proven or suspected carcinogens (such as
nitrous acid, hydroxylamine, and ethylmethane
sulfonate, EMS), Adding alkyl group to hydrogenbonding oxygen of G or T, resulting in G-T mispairing

G-C ---> G*T --->A-T

T-A --->T*-G ---> CG

Light Agent
Justafewtypesofdamageisrepairedvia
simplereversalofthechemicalchange
UVlightinduceddimers
Methylationofbases
Ethylationofbases
LargechemicalgroupsaddedtotheDNA

Mostotherdamagerequireothersystems

06_24_radiation.jpg

Randomphotonsofultraviolet(UV)lightinduceaberrantbonding
betweenneighbouringpyrimidines(thymine&cytosine)baseson
thesamestrandofDNA.Thewillpreventthereplicationmachine
fromduplicatingtheDNA.Thecellwilldie!

Thistypeofdefectcanbereadilyreversedbyaprocesscalled
photoreactivation.Visiblelightenergyisusedtoreversethedefect(in
bacteria,yeasts,protists,someplants,andsomeanimalsbutNOTin
humans)

Base-analogue Agents
A base analogue is a substance
other than a standard nucleic acid
base that can be incorporated into a
DNA molecule by the normal
process of polymerization. Such a
substance must be able to pair with
the base on the complementary
strand being copies, or the 3'->5'
editing function will remove it.
For example, 5-bromouracil is an
analogue of thymine and might
cause an A-T to G-C transition
mutation.

Base Analogue

Intercalating Agents:
Intercalating agents: Substances whose
dimensions are roughly the same as those of a
purine-pyrimidine pair. In aqueous solutions,
these substances form stacked arrays, and are
also able to stack with a base-pair by insertion
between two base-pairs. This may result in
frameshift mutation.

Model of intercalating agent

induced mutagenesis

Other forms of DNA damage

DeaminationAnaminogroupofCytosine
isremovedandthebasebecomesUracil
DeaminationAnaminogroupofAdenine
isremovedandthebasebecomes
Hypoxanthine
DeaminationAnaminogroupofGuanine
isremovedandthebasebecomes
Hypoxanthine

And
Depurinationthebaseissimplyrippedout
oftheDNAmoleculeleavingagap(likea
missingtooth)

Molecularlevelview
Rememberthesearerandomevents

06_23_Depurination.jpg

DNAlevelviewofthesametwoeventsaslastslide

06_25_mutations.jpg

Metabolite Mutagens
Chemicals that are metabolized to
electrophilic reagents: Aflatoxins,
benzo[a]pyrene
A mutagen is a physical or chemical agent
that causes mutations to occurs.
Mutagenesis is the process of producing a
mutation.
Mutant refers to an organism or a gene that
is different from the normal or wild type.

Reversion and
the Ames test:
Mutants may have second
mutation and become wild
type again.
Reversion was used as a
means of detecting
mutagens and carcinogensthe Ames test

DNA Repair Mechanisms

(1) Repair by direct reversal: The simplest
mechanism. e.g. UV induced T-T dimer is
recognized by photolyase and is cleaved
into intact thymine (light dependent). This
is called photoactivation

Excision Repair
(2) Excision Repair:
The most
ubiquitous repair
mechanism, which
can deal with a
large variety of
structural defects
in DNA.

Recombinational Repair
(3) Recombinational repair (Postreplicational
repair): Occurs before excision repair has
happened or when excision repair can not
fix the problem

The SOS response

(4) The SOS response: The SOS response
system is only active in response to some
signal such as a blocked of replication fork.
In E. Coli, recA and lexA govern the
expression of a number of other genes
involved in DNA repair. This is an errorprone DNA repair mechanism and result in
higher than normal mutagenesis.

SOS DNA Repair

1.DNAdamage
2.RecAconvertedtoRecA*
3.RecA*facilitatedLexAselfcleavage
4.IncreasedsynthesisofSOSproteins
5.Errorpronerepairinduced
6.DNAdamagerepaired
7.RecA*returnedtoRecA
8.LexAnolongerselfcleaved
9.LexArepressedSOSgenes
10.LexArepresslexAgeneexpression

Type of Mutations(I)
I. Point mutation:
A. Base substitution
Change in DNA
Transition: One purine replaced by a different
purine;or one pyrimidine replaced by a
diferent pyrimidine
A G T C
Transversion: A purine replaced by a pyrimidine
or vice versa
A
T
C G

Type of Mutations (II)

Change in protein
1. Silent mutation: altered codon codes for
the same a.a. GAG (Glu) --->GAA (Glu)
2. Neutral mutation: altered codon codes for
functional similar a.a. GAG--->GAC (Asp)
3. Missense mutation: altered codon codes
for different dissimilar a.a.
GAG ---> AAG (Lys)
4. Nonsense mutation: altered codon
becsomes a stop codon
GAG ---> UAG (stop)

Type of Mutations (III)

B.Frameshiftmutation:additionordeletionofone
basepairresultinashiftofreadingframeand
alteraminoacidsequence
1.Wildtype:

ATGACCAGGTC
Met

Thr

Arg

2.Baseaddition: ATGACACAGGTC
Met

Thr

Gln

3.Basedeletion: ATGACAGGTC
Met

Thr

Gly

Val

Type of Mutations (IV)

II.Insertion
III.Deletion
IV.Translocation
V.Inversion

Sickle Cell Disease

Thisisaverygoodillustrationofthedevastating
effectsofeventinychangestotheDNA
RedBloodCells
Hemoglobin
Hasalargeproteincomponent
2betaglobinchains
Asinglebasechangesubstitutioncausesthedisease

06_19_sickle_cell.jpg

Cellular protection from DNA damage

Naturalerrors:polymerasebaseselection,
proofreading,mismatchrepair
Endogenous/exogenousDNAdamage:base
excisionrepair,nucleotideexcisionrepair,
(recombination,polymerasebypass)
Recombinationandpolymerasebypassdonot
removedamagebutremoveitsblockto
replication.Polymerasebypassisitselfoften
mutagenic.

Which is which?
Thecellhasabigproblemtoovercome
Howdoesittellwhichstrandcarriedthe
correctinformation?
Wethinkweknow

06_21_Errors corrected.jpg

Thecellhastopicktherightstrandtofixorelse

Correction mechanisms
Directreversalofdamage
Photoreactivation(bacteria,yeast,some
vertebratesnothumans)Twothymines
connectedtogetherbyUVlight.
ExcisionRepairremovalofdefective
DNA.Therearethreedistincttypes
1)baseexcision
2)nucleotideexcision
3)mismatchrepair

base-excision
PresenceoftheUracilinDNAisagreat
exampleofthistype
Specialenzymesreplacejustthedefective
base
1snipoutthedefectivebase
2cuttheDNAstrand
3Addfreshnucleotide
4Ligategap

nucleotide-excision
Sameaspreviousexceptthat
Itrecognizesmorevarietiesofdamage
RemovelargersegmentsofDNA(10100sof
bases)

mismatch repair
SpecialenzymesscantheDNAforbulky
alterationsintheDNAdoublehelix
Thesearenormallycausedbymismatched
bases
AG
AC
CT
TheseareexcisedandtheDNArepaired

Base excision repair (BER)

Majorpathwayforrepairofmodifiedbases,uracil
misincorporation,oxidativedamage
VariousDNAglycosylasesrecognizelesionand
removebaseatglycosidicbond,thereby
producinganabasicorAP(apurinic/
apyrimidinic)sitebybaseflippingout
OneofseveralAPendonucleasesincises
phosphodiesterasebackboneadjacenttoAPsite
APnucleotideremovedbyexonuclease/dRPase
andpatchrefilledbyDNAsynthesisandligation

Mechanism of BER

NH2
4

O
H2C
O

N
1

CH3

HN

O
H2 C
O

glycosidicbond

deoxycytosine

deoxyuracil

thymine

Types of lesions repaired by

BER
Oxidativelesions;8oxoG,highlymutagenic,

mispairswithA,producingGC>TA
transversionsexampleMutY,MutM=FpgfromE.
coli
Deoxyuracil:frommisincorporationofdUor
deaminationofdC>dU,exampleUng,uracilN
glycosylase
Variousalkylationproductse.g.3meA
Theselesionsarenotdistortinganddonotblock
DNApolymerases
Spontaneousdepurination(esp.G)yieldabasic
sitesthatarerepairedbysecondhalfofBER
pathway

Flipping out
mechanism

Mismatch repair (MMR)

DespiteextraordinaryfidelityofDNAsynthesis,errorsdo
persist
Sucherrorscanbedetectedandrepairedbythepost
replicationmismatchrepairsystem
Prokaryotesandeukaryotesuseasimilarmechanismwith
commonstructuralfeatures
DefectsinMMRelevatespontaneousmutationrates10
1000x
DefectsinMMRunderliehumanpredispositiontocolon
andothercancers(HNPCC)
MMRalsoprocessesmispairsthatresultfromheteroduplex
DNAformedduringgeneticrecombination:acttoexclude
homeologousrecombination

Mechanism of MMR
5'
3'

CH3

CH3

MutSMutLMutH
5'
3'

CH3

3'
5'

UvrD+ExoIorExoXorExoVII
5'
3'

CH3

CH3

PolIII+ligase
5'
3'

CH3

MutSMutLMutH

Initiation
CH3

5'
3'

Excision
3'
5'

5'
3'

3'

5'

5'

3'

CH3

CH3

3'
5'

UvrD+RecJorExoVII
CH3

CH3

3'
5'

PolIII+ligase

Resynthesis
CH3

3'
5'

CH3

CH3

3'
5'

Mechanism of MMR
5'
3'

CH3

CH3

MutSMutLMutH
5'
3'

CH3

3'
5'

UvrD+ExoIorExoXorExoVII
5'
3'

CH3

CH3

PolIII+ligase
5'
3'

CH3

MutSMutLMutH

Initiation
CH3

5'
3'

Excision
3'
5'

5'
3'

3'

5'

5'

3'

CH3

CH3

3'
5'

UvrD+RecJorExoVII
CH3

CH3

3'
5'

PolIII+ligase

Resynthesis
CH3

3'
5'

CH3

CH3

3'
5'

Basis of MMR recognition

MutSdimer(inyeast,Msh2/Msh3orMsh2/Msh6
heterodimer)
ByDNAbindingexptsinvitroandDNA
heteroduplexrepairexptsinvivo:MMRcan
recognizeallbasesubstitutionsexceptC:Cand
shortframeshiftloops<4bp
TransitionmispairsG:TandA:Candonebase
loopsareparticularlywellrecognized(theseare
alsothemostcommonpolymeraseerrors)

StructureofMutSboundtoDNA
60kinkinDNA
Widensminor
groove,narrows
majorgroove

Nucleotide excision repair (NER)

RecognizesbulkylesionsthatblockDNA
replication(i.e.lesionsproducedby
carcinogens)example,UVpyrimidine
photodimers
Commondistortioninhelix
Incisiononbothsidesoflesion
ShortpatchofDNAexcised,repairedby
repolymerizationandligation
InE.coli,mediatedbyUvrABCD
Manymoreproteinsinvolvedineukaryotes
Canbecoupledtotranscription(TCR,
transcriptioncoupledrepair)

Xeroderma pigmentosum

Autosomalrecessivemutationsinseveral
complementationgroups
Extremesensitivitytosunlight
Predispositiontoskincancer(meanageofskincancer=8
yrsvs.60fornormalpopulation)

Recognitionandbinding

UvrAactsasclassical
molecularmatchmaker

Incision

Nicksdelivered3
and5tolesionby
UvrBC

Excisionandrepair

Shortfragment
releasedby
helicaseaction

Human NER

Rad1/10

Rad2 inS.cerevisiae

Further references

Friedberg.DNArepairandmutagenesis.ASMPress,Washington,D.C.
*MartiTM,Kunz,C,FleckO.2002DNAmismatchrepairandmutation
avoidancepathways.J.Cell.Physiol.191:2841
*Harfe BD, Jinks-Robertson S. 2000 DNA mismatch repair and genetic
instability. Annu. Rev. Genet. 34: 359-399.
*Krokan, HE, Standal, R, Slupphaug, G. 1997 DNA glycosylases in the base
excision repair of DNA Biochem. J. 325: 1-16.
*De Laat, WL, Jaspers, NGJ, Hoeijmakers, JHJ. 1999 Molecular mechanism
of nucleotide excision repair. Genes Dev. 13: 768-785
Petit, C, Sancar, A. 1999 Nucleotide excision repair: from E. coli to man.
Biochimie 81: 15-25
*Goodman, MF, Tippin, B. 2000. Sloppier copier DNA polymerases involved
in genome repair. Curr. Opin. Genet. Dev. 10:162-168.
*Friedberg, EC, Wagner, R, Radman, M. Specialized DNA polymerases,
cellular survival and the genesis of mutations. Science 296: 1627-1630.
Goodman, MF 2002. Error-prone repair DNA polymerases in prokaryotes and
eukaryotes. Annu. Rev. Biochem. 71: 17-50

06_26_three steps.jpg

Basicmechanismis
thesameforall
threetypes
1) Removedamaged
region
2) ResynthesisDNA
3) Ligate

ThankYou