Lecture #9 | DNA Repair, Mutagenesis, and Human Disease

Which is more dangerous UV A, B or C

UV C shorter wavelengths are more dangerous

• in practicality: UV C is absorbed by Ozone

• UV B responsible for sun burns

• We are much more exposed to UV A so its more dangerous

What do different types of mutations suggest?

Different mutations are characterizes by different types of lesions and hence have different types of agents

• these are signatures in the DNA

DNA damaging agent determined the lesion, the lesion determined the repair process

Common features of DNA repair systems

• Highly conserved

• Often inducible (always present and go into action when needed)

• Repair systems are linked to other processes

  • replication, cell cycle, transcription

Common features of mechanisms

• recognition of damage

• excision

• resynthesis

• ligation

Enzymes: polymerase and lygase

#1 Mismatch repairs (MMR)

Lesion: DNA polymerase

Repair enzyme: MutS, MutL

Cancer: colon cancer

DNA Poly is highly efficient in proof reading but still makes some mistakes

• if the organism is deficient in proof reading, it will die early from many different types of cancer

Repair: Mismatches, small insertions, deletions

1. MutS homologs (6 types) bind to site of mismatch through forming heterodimers based on the mismatch

2. Recruits MutL enzyme which acts as a bridge between the MutS and DNA helicase as it keeps track of the parental strand and they will act together to cut

3. DNA helicase and exonuclease expand the incision forming a very large gap

4. Gap repaired by a polymerase and a ligase

#2 Base excision repair (BER)

Lesion: Damaged based through oxidation or alkylation

Repair: Glycosylases, AP endonuclease, lyase

• There are many glycosylases which depends on the type of lesions

Cancer: Not clear what cancers are created due to a deficiency in this system

Mechanism:

1. Improper base if there is deamination

2. DNA glycosylase removes modified base by cleaving glycosylic bond

3. And endonuclease classes the phosphodiester backbone

4. AP lyase removes the sugar phosphate

5. DNA polymerase fills the 1 base gap and ligase seals the nick

Types of lesions repaired by BER

1. Spontaneous damage: deamination and depurinaton

2. Alkylation

3. Oxidative damage

   1. 8-oxo-G

   2. thymine glycol

#3 Nucleotide excision repair (NER)

Lesion: Bulky adducts

Repair enzymes: XP protein including helicase

Cancer: Skin cancer

Note: caffein can cause this distortion

Mechanism:

1. Bulk adducts forms (UV dimers, BPDE, AFB1 adducts, crosslinks) causes a distortion in the DNA helix

2. XP-C recognizes the distortion in the double helix

3. A precision complex is formed

   1. TFIIH complex containing helicases XP- and XP-D melts 30 bp around the damage

   2. XPA confirms the presence of damage by probing for abnormal backbone structure

   3. RPA (replication protein A) stabilizes the open intermediate by binding to the undamaged strand

      

4. XP-F and XP-G are structure specific endonucleases that make dual incisions in the DNA

5. With the aid of proliferating cell nuclear antigen (PCNA), 24 to 32 new fragment containing the damage is released

#4 Transcription coupled repair

Lesions: modified bases, bulky adducts

Repair enzymes: CSA, CSB, XP proteins

Disease: Cockayne Syndrome

Basic features of TCR

1. Preferential repair of actively transcribed regions

2. Preferential repair of actively transcribed strand

3. Coupling factors that recognize stalled RNA polymerase

4. Proteins involved in Bothe DNA repair and transcription

   1. TFIIH

Mechanism of TCR

1. RNA polymerase II stalls at a lesion in the transcribed strand

2. Stalled Pol 11 recruits CSB and UVSSA

3. CSB recruits TFIIH, CSA, XPG (endonuclease) and the repair proteins

#5 Recombination repair: post replication | Double-strand break repair

Homologous recombination (few errors)

Lesion: repair of ds breaks (radiation damage)

Repair enzymes: BRCA 1/2, Rad51, ATM

Cancer/Disease: Breast cancer

Non-homologous end joining (error prone)

Repair enzymes: Ku proteins, DNA-PK

Cancer: various

Recombination repair mechanism for ds break

1. Many proteins are involved in the initial steps: ATM, BRCA, RPA

2. Exonucleases chew back the ends of the DNA leaving ss overhangs

3. Rad51 forms nucleoprotein filaments starting in the ss regions and extending into ds regions

4. Rad proteins search for homology between the damaged strand and the other chromosomal allele

5. Strand invasion and elongation by DNA polymerase creating a new homologous strand

6. The newly synthesized strand crosses back over the original duplex and base pairs with the ds

7. DNA polymerase uses the newly synthesized strand as template and fills in the gap. Ligase seals the nicks

Non-homologous end-joining

Does not involve recombination

• Kinase and KU80 and KU70 recognize ends of DNA and also play a role in VDJ doing in the immune system