7L10 DNA Damage & Repair

mutation

nonsense, missense, silent, frameshift

in-frame deletion

delete a multiple of 3 so no shift frame, but still bad

example: UUC in CFTR causes cystic fibrosis

DNA damage

spontaneous, environmental, carcinogenic

Ames Test

determines if compound is a mutagen,

uses salmonella typhimurium with inactive histidine biosynthetic pathway;

if a cell grows, it must’ve mutated to allow it to grow in the deadly medium

mutagens v carcinogens

cause change in DNA sequences;

cause cancer

deamination

C > U, 5MC > Thymine, Adenine > HypoX, G > X

deaminating chemicals

sodium nitrate and nitrite

cured meats, tobacco, pesticides, leafy veggies

most common DNA damage

single strand breaks, depurination

how does a mutation become cancer

when repair systems replace it wrongly

5MCs are found in

CpG islands, hypermethylation silences genes. have lots of mutations, c an be accidentally activated

oxidation

cellular respiration makes ROS that insert themself into genes

ROS

reactive oxygen species,

free radical hydroxide > G or T

sometimes C or A

sometimes single strand breaks

depurination

removes base from nucleotide, forms abasic site

abasic site

site with no base;

apurinic site AP site

alkylation

agents covalently modify bases in DNA

spontaneous SAM of G > 7MG

like sulfur gas

distorts double helix

sulfur mustard

inserts itself between DNA strands, links them together so they cant transcribe

thymine dimers

from uv radiation

forms cyclobutane ring, distorts DNA structure

DNA strand breaks

PDE bond broken

ionizing radiation

single strand, double strand (may be staggered)

damaged bases prevent DNA ligase

ionizing radiation

high energy, breaks covalent bonds;

cosmic rays, x rays, radioactive materials

repair systems

all cells have multiple:

mismatch repair, base-excision repair, NT-excision repair, direct repair

cancer is from accumulation of mutations

DNA repair pathway

1. Recognition of the lesion.

2. Excision of the lesion.

3. Resynthesis of the DNA.

4. Ligation of loose ends

mismatch repair

follows replication fork and checks work right after replication (hemi methylated DNA)

MutL-MutS complex recognizes mismatch, binds, recruits MutH

MutH identifies methylated sites in parental DNA, binds, and uses methylation status to make ss break in new strand

removal of mismatch

exonuclease digests from nick through mismatch

DNA pol 3 fills gap

DNA ligase seals the nick

base excision repair

detects damage bases, depurination, and ss breaks

DNA glycosylase cleaves bond to AP site

AP Endonuclease repairs with single strand break

DNA pol 1 makes new DNA

DNA ligase seals it

DNA glycosylase

cleaves glycosidic bond making AP abasic site

AP endonuclease

breaks PDE bond to help remove damage

DNA pol 1

has 5>3 exonuclease, high fidelity, replaces the damage

DNA ligase seals it

nucleotide excision repair

done if the entire nucleotide is problematic (causes DNA distortion)

is coupled with transcription

1. detection by excinuclease

2. helicase excises damaged DNA leaves gap

3. DNA pol 1 / DNA pol E fills gap

4. DNA ligase seals nick

excinuclease

Hlyzes 2 PDE bonds on either side of distortion

UvrABC excinuclease prokaryotes

XP excinuclease eukaryotes, has XPA-XPG

NER helicase

prokaryotes = UvrD helicase

eukaryotes = TF2H

non-homologous end joining

predominant mechanism in G0 and G1 for ds break

error prone repair (introduces mutations)

1. Ku70/80 complex binds loose ends

2. PKcs bridges broken ends with other factors

3. micro-homology (sticky ends)

   1. exonuclease removes bases

   2. DNA pol mu and lamda add bases (error prone terminal transferases)

   3. artemis endonuclease removes overhanging flaps

error prone terminal transferases

no template, just add random bases to repair DNA break

homologous recombination

no errors, repairs ds breaks, only in S or G2 phases

on broken chromosome, grab its partner and use the partner as template

BRCA genes involved

p53

activates genes involved in cell-cycle arrest, DNA repair, and apoptosis

“guardian of the genome”

kinases Plate where DNA damage, inc stability

mmr v ber v ner v nhej v hr

• mmr = mismatch/small insertion. immediate. detects dna distortion, removes and remakes one strand

• ber = small base damage (deam, ox, alkyl). dna glycosylase removes, ap endoN cuts backbone, DNA poly and ligase fix

• ner = bulky lesions (dimers). cuts nucleotides, DNA poly and ligase fill gap

• nhej = ds breaks in G1. Ku70/80 bind ends, bring near, ligase together (makes error)

• hr = ds breaks in G2/S. sister chromatid is used as template