When is direct reversal of damage used?
How is each accomplished?
- Ligation of break in phosphodiester backbone = DNA ligase
- Repair of O6-methylguanosine = MGMT
Types of DNA repair
- Excision Repair
i. Nucleotide excision repair (NER)
ii. Base excision repair (BER)
iii. Mismatch Repair (MMR)
- Direct Reversal of Damage
- Lesion Bypass
- Double-Stranded DNA breaks
i. Non-homologous end joining(NHEJ)
ii. Homologous recombination
In general terms, what steps are taken in excision repair?
- excision at the mutation site
- removal of mismatched or damaged nucleotide(s)
- addition of new DNA to fill the gap (use other strand as template)
- ligation of newly synthesized segment by DNA ligase
What sort of lesions does NER work on?
Lesions that distort the DNA structure and block DNA or RNA polymerase
- Thymine dimers (UV damage)
- Adducts (carcinogens)
How does NER begin?
distorted DNA recognized by multi protein complex with endonuclease activity
Once lesion is recognized, how does NER proceed?
- Endonuclease cuts damaged strand on BOTH sides of the lesion
- Removal of the oligonucleotide containing the lesion
- Helicase unwinds and releases the lesion-containing oligonucleotide
- DNA polymerase fills in the gap using other intact strand as template
- Ligase joins the strands
What are two different recognition pathways in NER?
- Global genome NER
2. Transcription-coupled NER
How does Global genome NER work?
recognizes lesions / distorted DNA in any part of the genome
How does Transcription-coupled NER work?
recognizes distorted DNA regions that are being actively transcribed
Name 3 different diseases caused by mutations in genes that mediate NER.
- Trichothiodystrophy (TTD)
- Xeroderma Pigmentosum (XP)
- Cockayne Syndrome (CS)
Explain Xeroderma Pigmentosum
Mutation in DNA repair system (TFIIH complex)
Photosensitivity - unable to repair UV damage (thymine dimers; pyridine dimers)
Give some symptoms of Cockayne Syndrome
Failure to grow / gain weight (short stature)
Microencephalopathy (cognition issues)
Underlying genetic problem in Cockayne syndrome?
mutations in the DNA repair system; specifically NER system
sensitive to UV damage (cannot repair)
cells accumulate mutations - premature aging / cell death
Give some symptoms of Trichothiodystrophy.
Brittle hair: lacks sulphur
Genetic factors of Trichothiodystrophy?
Repair by TFIIH complex is not functioning
Symptoms of Xeroderma Pigmentosum?
Genetic problem in Xeroderma Pigmentosum?
TFIIH not functioning correctly in DNA repair
What is the main function of TFIIH?
subunit CDK7 phosphorylates C-terminal end of RNA poll II
subunits XPB and XPD have helicase activity to create the replication bubble
What sort of lesions does Base Excision Repair (BER) work on?
lesions that are missed by NER
these lesions may not distort the DNA or block polymerase function
What enzymes do the recognition for BER?
Glycosylases (familly of enzymes)
-each is specific to a particular altered base
What does uracil glycosylase recognize?
U in place of C in DNA as a result of C deamination
(C deamination replaces C4 - NH2 with C4=O
amine to ketone)
for repair by BER
What does 5-methylcytosine-DNA glycosylase recognize?
C5 methylated cytosine
for repair by BER
How does a glycosylase function once it has recognized a lesion?
“Flips” the altered base out and excises the base
i.e. hydrolyzes the N-glycosidic bond (btwn sugar and base)
Removal of base produces an apurinic / apyrimidic (AP) site
Once glycosylase has removed a base, what happens next?
AP-specific endonuclease (and AP lyase) removes the AP site
Gap is filled by DNA polymerase
Nick is sealed by DNA ligase
When is MMR utilized?
Very shortly after replication when a single base mismatch occurs
What proteins recognize the lesion in MMR?
Bactiria - MutS and MutL
Eukaryote - MSH and MLH (H = homologue)
How do MutS/MutL or MSH/MLH recognize the new vs. old strand during MMR?
New strand is not yet methylated
Once the lesion is recognized, how does MMR proceed?
- Excision near the lesion site (one single cut) on the new strand
- Exonuclease activity chews up the lesion-containing oligonucleotide
- Helicase assists to open the DNA
- Polymerase fills the gap using old strand as template
- Ligase joins the two strands
Give an example of a hereditary disease caused by mutation in MMR repair machinery
hereditary non-polyposis colorectal cancer (HNPCC)