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9. Gøre rede for vigtigheden af DNA repair. Beskrive princippet i DNA repair ved base excision og nukleotid excision i prokaryote og eukaryote celler
Devlin, s.191
Devlin, s.194-6, fig.4.28, fig. 4.29
Stryer, s.770-3

The accuracy of DNA replication is very big, but not 100%.

DNA polymerase increases the replication accuracy by 2 processes. The initial selection brings the error rate down to 10^{- 4} and the proofreading down to 10 ^{- 9}.

Still, there is handful of errors introduced during each round of replication.   

The maintenance of the integrity of the genetic message is key to life. Consequently, all cells possess mechanisms to repair damaged DNA.

A difference should be made whether the base itself is damaged or if the wrong base is incorporated!!!

If the base itself is damagd, there are three pathways of repair:

• Direct pathway - fx. an enzyme binds to the distorted region (pyrine dimer) and uses light energy to cleave it into the original state

• Base-excision pathway

• Nucleotide excision pathway

The basic principle of excision-repair pathway is the following:

• Removal of the damaged nucleotide

• Leaving a gap in the DNA

• Re-synthesis using the genetic information on the opposite strand

• Ligation to restore continuity of the DNA

Base-excision repair

Used in removal of damaged bases (methylated, delaminated, oxidized). I will explain it stepwise:

1. Detection: The damaged DNA is detected by a DNA glycosylase. Binding of the glycosylase flips the mismatched base into the active site of the enzyme.
    

2. Excision: removal of the single base from the DNA backbone by a glycosylase that cuts the N-glycosidic bond between the sugar and the base. The sugar-phosphate backbone is not broken. An AP (abasic) site is created, that must be removed. The sugar-phosphate backbone is cleaved at 5` to the abasic site by an AP endonuclease, but the sugar is still attached with its 3` end to the next nucleotide. AP lyase cuts this bond and completely removes the sugar-phosphate component that was linked to the damaged base.
    

3. Re-synthesis: The gap is filled by DNA polymerase I (prokaryotes) or DNA polymerase b (eukaryotes)
    

4. The remaining nick is sealed by DNA-ligase.

Nucleotide excision repair

Acts on a variety of damages, typically involving large adducts or distortion of the double helical structure of the DNA.

Stepwise:

1. Detection: the damaged base is recognized by a DNA repair complex
    

2. Excision: the segment around the damage is excised by an enzyme complex that makes two nicks (breaks the sugar-phosphate backbone) in the same strand, one on each side of the damage. An oligonucleotide (27-29nt in humans, 12-13 in E.Coli) is released.
    

3. Re-synthesis: The gap is filled by DNA polymerase I (prokaryotes) or DNA polymerase b (eukaryotes). The 3`- end of the nicked strand is the primer, and the intact complementary strand is the template.
    

4. The remaining nick is sealed by DNA-ligase.
   
    

Mismatch Repair

Mismatches are not DNA-damage, there are no damaged or modified bases present, just the wrong one of the four bases is incorporated. Thus recognition of mismatches relies on the distortion of the double helical structure.

When a mismatch is recognized, the cell has a problem because both bases are normal; it does not know which one of them should be excised.  The base lying on the newly synthesised strand must be the one which is wrongly incorporated, therefore the repair system must figure out which one of the two strands is the daughter strand.

The brief time during which newly synthesized DNA is not methylated provides time within which the strand can be recognized. DNA in most organisms is methylated at specific positions. When the repair complex recognizes these non-methylated positions, it can remove the wrongly incorporated base by excision repair.

Fx.
Hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome) results from defective DNA mismatch repair. This is not a rare disease, ca. 1 in 200 people will develop this form of cancer.
Mutations in two genes, called hMSH2 and hMLH1, account for most cases of the hereditary predisposition to cancer. These two genes incode proteins that obviously participate in recognizing, binding and cleaving of the mismatched basepairs.
This leads to accumulation of mutations thoriught the genome. In time, genes important in cell proliferation become altered, resulting in onset of cancer.       

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