Lecture 4 : SSBR Protein linked DNA breaks and neurological disease

How does PARP respond to single-strand breaks?

PARP1 binds SSBs and synthesises PAR to recruit repair factors

What is PARP hyper-activation and its consequence?

Excessive PAR synthesis depletes NAD+/ATP, triggers cell death pathways (parthanatos), harming neurons

Why do SSBR mutations cause PARP hyperactivity?

Persistent SSBs keep PARP chronically activated since lesions are not resolved

Why are neurons especially vunerable to SSBR defects?

High oxidative metabolism and active transcription → many SSBs; post-mitotic (no replication based repair), limited regenerative capacity

How does oxidative stress in neurons contribute to DNA damage?

Reactive oxygen species create base damage and SSBs that require SSBR

Why must cancer drug choice consider SSBR mutations?

Some chemotherapies (e.g. topoisomerase poisons) increase SSB/DSB load and can exacerbate neurotoxicity in patients with SSBR defects

How can patients tolerate partial loss-of-function SSBR mutations?

Residual low activity, partial protein function, or redundant pathways can maintain viability early in life

What are genetic redundancies in DNA repair?

Alternate enzymes/pathways that process similar lesions (e.g. multiple end processing nucleases)

How do complementation assays identify redundancies?

Introduce candidate genes into mutant cells; restoration of phenotype indicates functional compensation

What is TTRAP and its enzymatic activity?

TTRAP (TDP2-related protein) processes abortive TOP2 5’ phosphotyrosyl adducts and can act on some TOP1-like substrates

Which topoisomerase adduct does TTRAP primarily remove?

TOP2 (5’-phosphotyrosyl) adducts

Can TTRAP act on TOP1 adducts?

It has some activity against TOP1-type lesions but is less specialised than TDP1

What disease is caused by TTRAP mutations?

Spinocerebellar ataxia, autosomal recessive 23 (SCAR23)

How does loss of TTRAP, lead to neurodegeneration?

Accumulation of unresolved topoisomerase adducts → persistent SSBs/DSBs, genomic instability, neuronal death

How are double-strand breaks measured in cells?

yH2AX immunostaining detects phosphorylated H2AX foci at DSB sites

What is yH2AX?

H2AX histone phosphorylated at aerine-139 near DSBs; marks sites for repair factor recruitment

What does increased yH2AX signal indicate?

Elevated DSBs or persistent repair intermediates; genome instability

How do PARP inhibitors affect cells with SSBR defects?

Can be synthetically lethal in cancer but may worsen neuronal death if PARP hyper-activation is already present - drug choice must be cautious

What experimental assays test SSBR capacity in patient cells?

Comet assays (alkaline), yH2AX staining, mas-spec for adducts, complementation with candidate genes

Why can developmental viability occur despite mutations causing adult neurodegeneration?

Development relies less on affected pathways or compensatory mechanisms operate during development; cumulative damage over time leads to late-onset degeneration

Why do SSBR mutations Kill neurons?

Persistent SSBs chronically activate PARP, deplete NAD+/energy, and cause cell death in non-dividing neurons with high oxidative /transcriptional stress

How can a viable human be born with neurodegenerative mutations?

Residual/abundant repair activity and lower early-life damage allow development; damage accumulates over time causing later degeneration

How do complementation assays work?

Introduce a wild-type gene into mutant cells; rescue of function indicates that gene complements the defect

What is TTRAP’s role?

Processes abortive TOP2 5’-phosphotyrosyl adducts (and some TOP1 lesions), facilitating repair and ligation

What is yH2AX and what does it indicate?

Phosphorylated H2AX marking DSB sites; increased yH2AX reflects DSB presence or unresolved DNA damage