Beruflich Dokumente
Kultur Dokumente
Endah Wulandari
Biokimia Farmasi (SMT Genap) 2014
DNA Damage
Mechanisms for maintaining genetic stability associated
with DNA replication in E. Coli
Machanism
Base pairing
~10-1 - 10-2
~10-5 - 10-6
~10-7
~10-10
Spontaneous alterations:
(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.
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
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.
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
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
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
ATGACCAGGTC
Met
Thr
Arg
2.Baseaddition: ATGACACAGGTC
Met
Thr
Gln
3.Basedeletion: ATGACAGGTC
Met
Thr
Gly
Val
06_19_sickle_cell.jpg
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
Mechanism of BER
NH2
4
O
H2C
O
N
1
CH3
HN
O
H2 C
O
glycosidicbond
deoxycytosine
deoxyuracil
thymine
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
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'
StructureofMutSboundtoDNA
60kinkinDNA
Widensminor
groove,narrows
majorgroove
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