DNA Repair

proofreading

done by DNA polymerase

most common mechanism to prevent errors during replication

detect errors (mismatch), removes bases (exonuclease activity)

destroy free radicals

SOD - superoxide dismutase remove the reactive oxygen species (ROS)

direct reversal of UV damage

CPD photolyase enzyme + visible light => reverse damage

if fails: nucleotide excision repair is used

if NER fails: SOS repair is used

if SOS fails → mutations: C → T transition

homology dependent repair

uses opposite strand of DNA to repair

removes and replaces bases

3 mechanisms:

1. base excision repair

2. nucleotide excision repair

3. post-replication repair: mismatch repair

base excision repair

fixes minor damage

changes that does not distort DNA structure

ex: depurination / depyrimidation / deamination

steps in base excision repair

1. Remove the mistake: done by DNA glycosylate. - that empty space is now called AP site

1. Cut DNA backbone to make space:

• in prok: dRpase = deoxyRibose phosphodiesterase

• in euk: AP endonuclease

1. Correction:

2. For short patch correction: DNA pol B (euk only)

3. For long patch correction: Flap endonuclease

1. DNA ligase seals up

nucleotide exicision repair

mainly repair UV damage (pyrimidine dimers T=T)

2 pathways:

1. global genomic repair

2. transcription coupled NER

global genomic repair

aka Global Genome NER

occur in silent parts of genome

1. XPC and XPE recognize T=T

2. recruit TF complex containing XPB and XPD to unzip the genes

3. RPA and endonucleases cut out the damage and remove

4. PCNA and DNA pol D/E fill the gap

5. ligase comes to seal up

slow process

transcription coupled NER

NER = Nucleotide-excision repair

occurs during RNA transcription

1. RNA polymerase recognizes BULKY DAMAGE that distorts the helix → stalled → transcription paused→ recruits CSA/CSB

2. recruit TF complex containing XPB and XPD to unzip the genes

3. RPA and endonucleases cut out the damage and remove

4. PCNA and DNA pol D/E fill the gap

5. ligase comes to seal up

faster than GGR

XPB, D = xeroderma pigmentosum proteins B and D

similarity in NER and GGR

XPB/XPD protein complex contains helices to unzip the genes

recruit repair complex:

• RPA (replication protein A): binds and stablizes single strand DNA

• endonuclease: cut the damaged strand, remove adjacent nucleotides

• PCNA and DNA pol D and E are recruited

• New DNA strand is made from the single strand template

• DNA Ligase I seals up

Xeroderma pigmentosum

mutations in XPB and XPD

UV expo results in skin cancers

abnormalities of eye lid, conjunctiva and cornea

neurological deficit

Cockayne Syndrome

mutations in CSA/CSB

aka dwarfism-retina atrophy-deafness syndrome

RNA Pol stalls without repair → apoptosis

Phenotype:

• reduced cell prolif

• short stature

• premature aging

• pigmentary defects in retina

• neurological impairment

mismatch repair

• MutS regconizes mispair and bind to it

recruits MutH (in prok only) and MutL proteins

• Euk use PCNA to phan biet parent vs daughter strand

PCNA-MutL nicks daughter strand

• prok uses MutH to nick daughter strand

• DNA unwound → new strand error is removed

• DNA Pol D fills the gap

• DNA Ligase seals up

Hereditary non-polyposis colorectal cancer

predisposition to colon cancer

early age onset

defects in mismatch repair → defect in coding of MutS and MutL

SOS system

last resort option for repair

bypasses unprepared DNA damage at stalled rep forks

Non homologous end joining

repairs double stranded breaks

last resort

permits replication without correcting errors

always result in indels, sometimes translocation or inversions

can cause chromosomal rearrangements

trans-lesion synthesis definition

the cell uses trans-lesion synthesis when NER fails

Polymerase III stalls at the damaged strand, bypass Pol comes to replaces poly III and continued on the replication

allows DNA rep across the damaged pair without correction

save cell from apoptosis

trans-lesion synthesis mode of action

• DNA Pol III (D/E in euk) recognizes damage and stalls

• “bypass polymerase” recruited by the clamp (beta clamp in prok and PCNA in euk) and replaces Polymerase at work

• “bypass polymerase” replicates DNA across the site of damage without correcting damage

• “bypass polymerase” replaces by Pol III or whichever Pol that was there before

• DNA rep continues

Nonhomologous end joining mode of action

• Ku70/80 heterodimers bind to broken, double stranded DNA: stabilize the broken ends, recruits others for repair

• repair complex assembles:

  • DNA protein kinase

  • Artemis

  • XRCC4

  • NHEJ1

  • DNA ligase IV

• Process:

  • trim ends of DNA

  • Ligase seals up

NHEJ cons

no proofreading

no mechanism to match the strands if more than one break

always generates INDEL mutations

may cause translocations and inversions

inhibited at telomeres

homologous recombination

double strand breaks repair - high fdidelity

nucleases expose 3’ end of of break

3’ end invades homologous strand to form displacement loop

• DSBR: double D-loop and synthesis from both strands

• SDSA: helices kicks out invading strand

Type of AA substitution

conservative: minimal effects on protein and structure

non-conservative: major effects

mechanism of NHEJ

• Ku70/80 heterodimers bind and stabilize broken DNA ends

• repair complex assembles

• trim ends and ligates DNA together