TDP1 deficiency and genomic instability in non-replicating cells

Salimbeni, Simona (2020) TDP1 deficiency and genomic instability in non-replicating cells, [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Biologia cellulare e molecolare, 32 Ciclo. DOI 10.48676/unibo/amsdottorato/9499.
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SCAN1 is a rare recessive neurodegenerative syndrome associated with cerebellar atrophy and peripheral neuropathy. It is caused by a homozygous mutation in the TDP1 gene. This results in a substitution of histidine for arginine-493 (H493R) in the catalytic site, leading to reduced TDP1 activity. TDP1 hydrolyzes the bond between a DNA 3’-end and a tyrosyl moiety within a trapped TOP1cc. TDP1 also processes other 3’-end-blocking lesions, including 3’-phosphoglycolates resulting from oxidation of DNA. However, how TDP1 H493R mutation promotes the SCAN1 phenotype is unclear. DSBs are the most harmful genomic lesions. Their defective repair can induce cell death, and they have been implicated in the pathogenesis of several human diseases, including neurodegenerative syndromes. We aim to investigate whether the SCAN1 phenotype could be related to an accumulation of DSBs in non-replicating cells harboring the H493R mutation of TDP1. We have generated U2OS cells homozygous for TDP1 H493R or TDP1 KO and WI38 hTERT TDP1 KO employing the CRISPR-Cas9 technique. We found that both TDP1 H493R and TDP1 KO cells accumulate endogenous DSBs, primarily in G1 phase of the cell cycle compared to S. A similar increase of DSBs was observed in quiescent WI38 hTERT cells siRNA for TDP1, suggesting the replication-independent nature of DSBs. Treatment of TDP1 H493R and KO cells with camptothecin further suggests that the accumulation of DSBs could be related to the defective removal of TOP1ccs. We found also that TDP1 deficiency modulated R-loop levels at some gene loci, raising the possibility of their implication in DSB formation. Analysis of DSB repair following camptothecin treatment revealed that both TDP1 H493R and KO cells were defective in the repair of DSBs in G1 but not in S, with TDP1 H493R having the most pronounced effect. Together, our results provide insights into the etiology of the SCAN1 neurodegenerative syndrome.

Tipologia del documento
Tesi di dottorato
Salimbeni, Simona
Dottorato di ricerca
Settore disciplinare
Settore concorsuale
Parole chiave
TDP1, DNA damage, Transcription, R-loops, SCAN1 syndrome, Topoisomerase I
Data di discussione
29 Giugno 2020

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