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• Carcinogens– Carcinogens – agent that inducing
mutation of DNA leading to cancer– Procarcinogens – are not carcinog
ens, but can be activated by metabolic processes
Ames testHis- strain of bacteria Salmonea tyrphimuriumHis- can not synthesize HisMutagenesis indicates reversion of strain to His+
Cells of S. tyrphimurium is spread on culture plate lacking his in the presence of tested mutagent, growing cells indicated reversion to His+ strain.
• UV, short wave length (254nm)– Covalently link adjacent pyri
midines along one side– cis-syn-cyclobutane pyrimidi
nes dimers– Thymine or cytosine dimers– Formation of dimer distorts b
ase pairing structure=> effect replication and transcription
DNA damage• oxidative deamination: spontaneous reaction at 37oC
N
NN
N
O
NH2
H
H
N
NN
N
O
O
H
H
Guanine Xantine
Reaction with alkylation agents
-Addition of methyl group to bases- S-adenosylmethionine
- some agents used in chemotherapy
N
N
NN
O
NH2
HH
N
N
NN
O
NH2
H
CH3
Cytosine
Thymine
Alkylation agent
Alkylation of guanine
O6-methylguanine
• Reaction with ROS;– Number of mutations increase with age
N
NN
N
O
NH2
H
Hguanine
Depurination – cleavage of glycosyl bond results into removal of base - abasic sites, apurinic sites
DNA glycosylases remove altered bases
• Deamination of cytosine, particularly 5-methyl cytosine leaves uracil (thymine if 5methyl cytosine) in the DNA. Uracil would pair as thymine during replication and thus cause a mutation. Uracil-N-glycosylase removes uracil from DNA. An endonuclease then cleaves the backbone at that site, creating a substrate for NER
• Nucleotide excision repairVariety of damages, includes pyrimidine dimers, chemotherapic agents as cis platine
Repair mechanism involves removal of several nucleotides
E. coli: UvrABC endonuclease complex
UvrA – recognize helix distortion UvrB – separation of DNA strands (helicase activityUvrC – catalyses dual cleavageUvrD – exonuclease activity removes oligonucleotidePol I fills the gapDNA ligase – couple the nick
Mismatch repair corrects the unmethylated strand
What happens if the “old” strand needs repair?eg 5-methyl cytosine>deamination to 5-methyl uracil (=thymine!). In E. coli a small fraction ofC is 5-methylated and these are “hot-spots” forspontaneous mutation. This implies that 5-methyl cytosine is frequently either not repaired or ismistakenly repaired on the “wrong” strand.
Mis-paired bases
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