Bioc 302 Dna Damage And Repair

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Codon

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a nucleotide triplet in the DNA sequence that codes for a specific aa residue (or stop signal)

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open reading frame (ORF)

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a group of contiguous, non-overlapping nucleotide codons in a DNA or RNA sequence that begin w a start codon and do not include any stop codons

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mutation

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a permanent, transmissible change in the DNA (usually a single gene)

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mutagen

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a substance that causes an increase in the rate at which mutations occur

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carcinogen

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a substance that causes cancer
-most carcinogens (>90%) are mutagens

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transition mutation

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exchange of a purine w a purine or a pyrimidine w a pyrimidine base
-more common than transversion
-often the result of tautomeric shifts

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transversion mutations

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exchange of a purine w a pyrimidine base and vice versa
-more difficult to happen

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DNA damage

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1. polymerase errors
2. chemical rxns
3. radiation

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polymerase errors

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-limited by proofreading, but errors in pairing and additions/deletions do occur
-can be due to incorrect dNTPs, such as dUTP and 8-oxo-dGTP being incorporated

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chemical rxns to DNA

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-hydrolysis (mainly purines) and deamination (dC, dG, dA)
-oxidation
-alkylation by electrophilic reactants from exogenous/endogenous chemicals

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radiation to DNA

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UV
X-rays

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Depurination

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the N-glycosyl bond between the base and the pentose can undergo hydrolysis

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Base deamination

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nucleotide bases can undergo spontaneous loss of their exocyclic amino groups (=deaminatino)

eg. cytosine --> uracil (if taken into replication, get transition mutation)

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8-oxoG base pairs

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-can pair w C or A (can flip between syn and anti)
-results in transversion mutation

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5-Br-Uracil

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-can pair w A or G
-results in transition mutation

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oxidative damage

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-reactive oxygen species (ROS): OH*, H2O2, O2-
-variety of base lesions produced (typically small damage can potentially fit in polymerase active site, like mispair)

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alkylation damage

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7-position of guanine is most nucleophlic (grab methyl from alkylating agent)
-but all heteroatoms are potential targets based upon the chemical nature of the electrophile and its interaction w the DNA

-alkylation induces tautomerization and changes Watson-Crik H-bonding face

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Bulky Adducts (BaP)

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-inert Bap is converted to the reactive epoxide in an attempt by the body to make it hydrophilic so it can be excreted
-the epoxide is an electrophile that will react w nucleophiles on DNA

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adducts from UV radiation

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-DNA photoproducts formed between adjacent pyrimidines
-base substitutions are TT, TC, CT, CC sequences (w UV radiation, they link covalently)

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mutational hot spots on E coli chromosome have been shown to contain 5-methylcytosine. Why?

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base deamination
-if deaminate, then get thymine (pairs w A and results in transition mutation)

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in eukaryotic cells, a specific triphosphate cleaves 8-oxo-dGTP to oxo-dGMP + PPi
what is the advantage of this rxn?

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inactivation
-if monophosphate instead of triphosphate, it cannot go into replication (saves it)

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mismatch repair system

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E coli contains methylation system that methylates adenine bases in GATC sequences (palindromic)
-both strands will be methylated at least once every ~1kbp
-before replication, both strands methylated
-immediately after replication, the new strand will not be methylated, as the methylation system has not had time to act (DNA will be hemimethylated)
-during this delay, the mismatch repair system has the opportunity to recognize mismatches and repair the nonmethylated (new) strand for excision

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mismatch repair system steps

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1. the MutL/S proteins bind the mismatch
2. MutH binds to complex and allows scanning to the nearest GATC; it cuts the strand w the non-methylated GATC (MutH requires ATP)
3. after incision, the combined action of a helicase and an exonuclease degrades the strand up to the mismatch
4. repair synthesis by DNA Pol III

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base-excision repair system

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-bases that have been damaged or modified are removed by DNA glycosylases
-they catalyze the hydrolysis of the glycosidic linkage between the deoxyribose and the base

eg. uracil glycosylase removes the deamination product of cytosine

each glycosylase is specific for the type of base it removes

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base-excision repair system steps

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1. a DNA glycosylase recognizes a damaged base and cleaves between the base & deoxyribose in the backbone (remove damaged base)
2. the abasic AP (apurinic/apyrimidinic) site is recognized by enzyme AP endonuclease which will cleave the DNA at a point near the lesion
3. DNA Pol I can catalyze repair synthesis (nick translation) through the AP site
4. DNA ligase seals the nick

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nucleotide-excision repair system

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many types of lesions cause distortions in the double helical structure
-most common are pyrimidine dimers (caused by UV irradiation)
-non-specific for damage, as long as it is big and bulky

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nucleotide-excision repair system steps

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1. a specialized DNA exinuclease recognizes the distortion and cuts the DNA strand before and after the lesion
2. DNA helicase removes the fragment containing the lesion
3. the gap is repaired by DNA polymerase
4. DNA ligase seals the nick

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difference between nucleotide-excision repair system in prokaryotes and eukaryotes

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prokaryotes cut out smaller piece of DNA (13NT)

eukaryotes cut out bigger piece (29NT)

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direct repair

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repair certain common lesions in DNA without removing bases or nucleotides
-the bases are converted back to their original form while still part of the double helix

eg. photolyase
eg. O6-methylguanine-DNA methyltransferase

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photolyase

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*not in humans
-repair of pyrimidine dimers (damage due to UV radiation)
-uses light to fix linkages (does not cost the cell anything)
-radical destabilizes and breaks linkage
-uses FADH- to transfer radical

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O6-methylguanine-DNA methyltransferase

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-transfers the methyl group from O6-methylguanine to a Cys on the protein
-this inactivates the protein (suicide enzyme)
-inactive form w methyl serves as transcription factor to make more protein

O6-methylguanine pairs w T so if not repaired, will result in a transition mutation)