Cooperazione tra diversi sistemi di riparazione nella rimozione del danno indotto al DNA dalle radiazioni ultraviolette
Author(s)
Filippi, Silvia
Date Issued
October 3, 2008
Type
Doctoral Thesis
Abstract
During the cellular life, the DNA is generally exposed to the mutagen agents, that they can divided by theirs nature (physics o chemistry), their origin (endogenous and esogenous) o by the different DNA damage ( base modification, covalent link, break of elix…)
The DNA damage, if left unrepaired, could compromise the vital processes cellular, for example, transcription, DNA replication and cromosomic segregation and became cancer and cellular senescence.
The repair system perform a fundamental role in genome maintenance operating a secondby the type of lesions and of the cell cycle phase.
For example, the Nucleotide excision repair (NER) deals with the wide class of helix-distorting lesions, caused by UV-light o chemical agent. They is divided into two sub-pathway: Transcription coupled repair, focuses on damage present on the transcriptional region of the genoma, and Global genome repair surveys the entire genome for distorting injuri.
The importance of this repair system is demonstrated by the existence of two syndrome.
Patients affect from genetic syndromes, like Xeroderma Pigmentosum and Cockayn syndrome, showed the importance of this repair pathway. Infect cells derived from this patients have got an high sensitivity to UV-light. The highest DNA damage induced from UV-light are CPDs, 6-4PP and the inter-strand cross-links (ICLs). This type of DNA damage caused both DNA transcription arrest and replicativa fork arrest.
In our preliminary study we have made experiments to understand the importance both of nucleotide excision repair, and homologues recombination pathway, in the resumption of replicativa fork arrest done to UV-light.
We used isogenetic Chinese Hamster cells line defective respectively in NER and HR pathways, UV4 and irs1SF. Our data showed that DNA damage induced from UV-light caused replicativa fork arrest. This arrest induced apoptosis and the activation of both HR and transletion DNA synthesis repair pathways.
The co-treatment with caffeine induced an enhanced in the frequencies of SCE and a decries in the presences of base mutations. These data showed that the repair pathway used to remove DNA damage caused from co-treatment with caffeine and UV-light are homologous recombination more then TLS. Therefore the biology effect of the recombinational repair is the induction of chromosomal aberrations, the enhanced in the frequencies of SCE and the replicativa fork retrogression, whereas the TLS activation caused base mutations.
In the successive experiments we studied the relevance of photo-lesions done to ICLs, differences from CPDs and 6-4 PP, and the induction of chromosome instability. We used cells derived from patients affect to two different genetic syndromes, Cockayne syndrome (CS) and Xeroderma Pigmentosum (XP). Cockayne syndrome is caused by mutation in the CSA or CSB genes. The functionality of this two proteins is bind to the repair of pyrimidine dimer induced from UV-light in DNA transcriptional strand, transcription-coupled repair (TCR).
The Xeroderma Pigmentosum disorder arises from mutations in one of seven genes (XPA-XPG). The functionality of this seven proteins is bind to the repair UV-light lesions in all the genome, global genome repair (GGR).
The two syndromes patients showed high sensibility to sun radiations, neurodegenerations and premature ageing. Whereas XP patients showed a predisposition to skin cancer, this is not observed in CS patients.
We used isogenetic Chinese Hamster cells line to study the effect of ICLs in the chromosomal instability caused from UV-light. Precedents study showed an high induction of chromosomal aberration after UV irradiation in UV61 cell line, Cockayne like cell line, in compare to AA8 cell line, parental cell line.
These two cells line are different in the ability of repair CPDs lesions in the DNA transcriptional strand, infect the AA8 cell line are proficient, whereas the UV61 cell line are deficient in this repair pathway. Both the cells line are enable to repair the CPDs lesions in non transcriptional genome regions because they have a mutation in the p48 gene. Along both AA8 and UV61 cells line are abele to repair the 6-4 PP in all the genome.
To examine this idea we tested cells line deficient in NER-TCR pathway with mitomycine C, an agent that induced ICLs. We employed three different cells line, AA8, UV5 and UV61, respectively wild type, NER deficient, TCR deficient. We made cytogenetic (Hams test, chromosomal aberrations, SCEs) and biomoleculary (comet assay, western blotting) test. Our data showed that NER-TCR attend to the repair of ICLs, that there is a direct or indirect involvement of CSB protein in the activation of HR.
This results propose a synergic action of transcription couple repair and homologous repair during the inter-strand cross links repair.
Additional information
Dottorato di ricerca in Biologia e biochimica
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