DNA mutation & repair part 2

it may not have a drastic effect as not 3 (or a multiple of 3) nucleotides removed or added (frame shift)

• promoters or regulatory sequences
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• regulatory sequences
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• the intron not being removed
• the exon being spliced out
• a cryptic splice site being used instead
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• act directly on the damaged nucleotides, returning them back to their original structure without replacing the original bases
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involves excision of a damaged base and a segment of the polynucleotide containing a damaged site, followed by resynthesis of the correct nucleotide sequence by a DNA polymerase

• 2 groups of excision repair: base excision repair & nucleotide excision repair
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• involves excising a stretch of one strand of DNA containing the wrong nucleotide and then repairing by filling the gap
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At the point of break, deletion of the bases should be prevented. Correct ends should be joined. (If 2 chromosomes are broken, then the correct pairs must be brought together.)

2 distinct pathways: homologous recombination & nonhomologous end-joining (NHEJ)

copies across the damaged area (is a form of tolerating the damage while the repair is not possible)

direct repair

• phosphodiester (usually due to the damaging effects of ionizing radiation);
• DNA ligase (if there is no damage to the 5'-phosphate and 3'-OH groups of the nucleotides in either side of the nick)
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enzymatic transfer of alkyl group from the base to the polypeptide chain of the enzyme

• Ada enzyme of e.coli: removes alkyl groups attached to the oxygen groups at positions 4 & 6 of thymine & guanine, respectively
• Human MGMT: only removes alkyl groups from position 6 of guanine
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photoreactivation (a light-dependent direct system)

• In e.coli, DNA photolyase binds to cyclobutyl dimers & is stimulated by light, changing the cyclobutyl dimer to 2 thymines
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• 1. cleavage of the b-N-glycosidic bond by DNA glycosylase (creation of an AP site, or baseless site)
• 2. conversion of AP site to a single nucleotide gap by an AP endonuclease
• 3. filling the single nucleotide gap by a DNA polymerase using base pairing with the undamaged base in the other strand of DNA
• 4. restoration of phosphodiester bond by a ligase
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removed the damaged bases. Others remove normal but mismatch bases.

• Glycosylases scan DNA along the minor grooves until a damage is detected.
• The damaged base is flipped out to its active site & projects out of the DNA where it sits into a pocket of the enzyme.
• The enzyme catalyzes the removal of the base.

• 1. Uracil is removed by uracil glycosylase, which generates an AP site
• 2. AP endonuclease generates a gap in the strand
• 3. 3'OH is made
• 4. DNA polymerase fills the gap

• An A pairs with Oxo-G. Then a fail-safe glycosylase acts and removes the A (Although it is not damaged, it's incorrect.) allowing it to be replaced by C.
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• T is removed. (The assumption is T is the wrong base.)
• Methylcytosince naturally can be deaminated, so it becomes a T (mispaired with G)
• Thus, any T:G is assumed to be the result of this deamination, so it's the T that is removed, not the G.

distortion in the structure of DNA

Nucleotide excision repair is able to deal with more broad and extreme forms of damage (compared to base excision repair) such as intrastrand cross-links, attachment of large chemical groups, and correcting cyclobutyl dimers by a dark repair process.

• a segment of the strand of DNA that contains the damaged nucleotides;
• generates a gap in the damaged strand. The generated gap is replaced with new DNA by a DNA polymerase that uses the other strand (undamaged strand) as the template.

it is not preceded by selective base removal & a longer stretch of polynucleotide is cut out

• A complex of 2 UvrA & 2 UvrB scan the DNA for damage. UvrAs detect the distortion & UvrB melt the DNA.
• UvrCs are recruited & make 2 incisions, one down the 3' side & one up the 5' side
• The damage containing DNA strand is detached by helicase activity of UvrD.
• The generated gap is filled by polymerization activity of DNA pol I and then phosphodiester bonds are restored by a DNA ligase.

• RNA polymerase stalls as it reaches the DNA lesion during the transcription.
• Subunits of TFIIH with helicase activity (XPA & XPD) are responsible for melting the double strand DNA with lesion.
• The rest of the steps of nucleotide excision repair continue the process of repairing the lesion.

• The older strand is methylated so the new strand can be recognized. Errors of replication is corrected. (bases that are mismatched). The new strand is repaired.
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the kink they introduce in the structure of DNA

• MutS binds to the mismatch base.
• MutL is recruited → MutH is recruited.
• MutH binds to unmethylated 5'-GATC-3' sequences.
• MutH makes an incision in the new strand.
• A DNA helicase unwinds the new strand beyond the point of mismatch.
• An exonuclease removes the displaced strand
• DNA polymerase fills the gap

• The exposed single strand is coated with PARP1 proteins, which protects this intact strand from breaking and & prevents it from participating in unwanted recombination events.
• The break is then filled in by the enzymes involved in the excision repair pathways
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ionizing radiation & some chemical mutagens & can also occur during DNA replication at the fork of replication.

Double-strand break is the worse type of DNA damage & can have drastic effects on the cell

• Ku proteins (Ku70 & 80), one copy attaches to each broken DNA end.
• Individual Ku proteins have an affinity for each other, which brings the 2 broken ends of the DNA molecule into proximity.
• Ku binds DNA-PKCs protein kinase, which activates Artemis (exo/endonuclease) → processes the broken ends → prepares for ligation

• the undamaged sister chromosome;
• the DNA with both strand damage

• One strand in each half the broken DNA is shortened, so each end now has a 3' overhang.
• One of these overhangs invades the homologous DNA molecule (the intact homologous chromosome)
• Completion of the heteroduplex by extension of strands of the partner that suffered the double-strand cut and using the equivalent regions of the uncut partner as the templates
• cleavage of the 2 DNA molecules
• mismatch repair