DNA damage and repair

why can DNA damage result in cell death?

transcription and replication can be blocked, cell cycle stopped→

what do mutations arise from?

-errors replication of undamaged DNA

-errors replication of damaged DNA

-inaccurate repair damaged DNA

what are most mutations from tho?

inaccurate repair of damaged DNA

how much less do mutations form in sperm cells? (compared to somatic cells)

10x lower

what are the 2 ways DNA damage can occur?

spontaneously since DNA is chemical (e.g. oxidative), external agents (mutagens)

what are the 3 spontaneous ways DNA can be damaged?

chemical: deamination of cytosine, depurination

damage from reactive oxygen species (H2O2 & free radicals)

whats this?

deamination of cytosine

whats this?

depurination

what can the deamination of C cause?

C-G can switch to A-T pairing (if not repaired)

what can the depurination cause?

apurinic sites block transcription & can block replication

what are the 4 ways ROS interact with DNA?

chemical modification of bases (oxidise), formation of cyclopurines (structural distortion), interstrand cross-links, strand breaks

what are the 3 ways DNA damage can be induced?

ionising radiation (x-rays), ultraviolet (UV) radiation, chemicals (e.g. polycyclic aromatic hydrocarbons)

whats one of the most main forms of radiation?

radon (more or less exposure depending on where you live)

how does ionising radiation fuck up your dna?

creates radicals

exposure of UV to DNA causes?

cyclobutane pyrimidine dimers (CPDs), (covalent bonds between adjacent pyrimidines), formation of 6-4 photoproducts

whats this

cyclobutane pyrimidine dimer

whats this?

6-4 photoproduct

why is it bad photoproducts cause structural distortions in DNA?

prevents normal passage of DNA polymerase, and rna polymerase

what kind of uv creates most to least amount of CPDs?

UVC (100x more than) UVB (1000x more than) UVA

what are the usually good DNA repair mechanisms?

base excision repair, nucleotide excision repair, mismatch repair, homology directed repair

what are the error-prone DNA repair mechanisms?

non-homologous end joining, translesion sythesis (bypass damage)

how is cytosine deamination repaired?

base excision repair

how is cytosine depurination repaired?

base excision repair

how is base damage from ROS repaired?

base excision repair

how is interstrand crosslink repaired?

interstrand crosslink repair

how is interstrand crosslink caused?

some chemotherapy drugs

how is cyclobutane pyrimidine dimer repaired?

nucleotide excision repair

how is adduct repaired?

nucleotide excision repair

how is an adduct to DNA caused?

chemicals including PAHs

how is a double stranded break repaired?

homology-directed repair, non-homologous end joining

how does base excision repair work?

damage specific glycosylase enzymes slide along DNA , recognise and excise damaged bases → creates apurinic and apyrimidic sites

apurinic/ apyrimidic site endonuclease cleaves phosphodiester bond 5’ to the AP site

in mammals -lyase activity of beta DNA polymerase may remove the deoxyribose and phosphate previously linked to damaged base

DNA polymerase b replaces missing nucleotide, DNA ligase seals the gap

how does nucleotide excision repair work?

recognition of damage and unwinding → cleavage at sites flanking damage → DNA polymerase synthesise new DNA → DNA ligase seals gaps

what are the 2 types of nucleotide excision repair?

template-strand and global genome

how does GG-NER recognise dsmage?

the structural distortion detected by protein XPC

what does XPC recruit to help it recognise CPDs

UV-DDB

how does TS-NER recognise damage?

RNA polymerase stalls at damaged site, proteins CSA and CSB are recruited, RNA polymerase backtracks, marking site and allowing NER machinery access

what happens next in nucleotide excision repair after site recognition?

helicase activity of TFIIH unwinds DNA near damage site,

damaged strand cleaved on both ends by XPG and XPF,

release of fragment containing damaged site, synthesis of DNA to fill gap,

synthesis of missing phosphodiester bond by DNA ligase

how does non-homologous end joining work?

ku protein bind to both ends, recruits DNA-protein kinase catalytic subunit- interactions keep them close,

ku recruits nucleases,

DNA polymerases & DNA ligases,

DNA ligases re-joins the broken ends

what are the consequences of DNA damage

-mutation

-ageing 

what is the evidence DNA damage causes ageing?

mutations and DNA damage accumulate with age

all inherited disorders that lead to premature ageing come from mutations in genes involved in DNA repair and maintenance

cancer patients cured w chemotherapy that damages DNA show signs of premature ageing

what causes xeroderma pigmentosum?

due to mutations that compromise global genome- NER

what causes Cockayne syndrome?

mutations that compromise transcription coupled NER

what are the symptoms of xerodermal pigmentosum?

-heightened sensitivity of skin and eyes to sunlight 

-increased risk of skin cancer

-neurological degeneration

what are the symptoms of Cockayne syndrome?

-extreme sensitivity of skin to sunlight

-progressive neurological degradation

-premature ageing (death of non-proliferating cells)

what is the mechanism of cockayne syndrome?

-RNA polymerase stalls at damaged site

-without functional CSB or CSA, RNA polymerase II remains stalled on the gene and fails to backtrack, making damaged region inaccessible to all repair mechanisms