Minimal toolkits for eukaryotic DNA repair

Kania, Daria (2021) Minimal toolkits for eukaryotic DNA repair. PhD thesis, UNSPECIFIED.

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Abstract

Delivering undamaged genetic material to the next generation is the primary aim of every living organism. Given the continual threat of DNA damage caused by endogenous and exogenous factors, a number of systems that continually detect, alert, and repair DNA damages, have evolved naturally in the whole spectrum of living organisms. The protozoan parasites are excellent examples of eukaryotes that show extreme adaptation to the environment they are exposed to. However, there is much to uncover about the DNA repair in these human parasites. This work presents here the overall picture emerging from my studies of DNA repair mechanisms in eukaryotic organisms with an emphasis on the proteins found in human-pathogenic parasites with a particular interest in G. intestinalis, T. vaginalis, T. brucei, and Leishmania species. In the first chapter of results, I demonstrate that a purified recombinant family 1-related uracil-DNA glycosylase from G. intestinalis (GiUDG) that lacks various residues that were thought to be essential for the activity of the previously characterised Family 1 UDGs is indeed an active uracil-DNA glycosylase. GiUDG remained active in the presence of Ugi. The site-directed mutagenesis studies revealed that many of the remaining amino acid residues that are conserved between GiUDG and other Family 1 enzymes could be mutated without affecting the enzyme’s activity. In the second chapter of results, I demonstrate that T. vaginalis possesses two active 3-alkyladenine DNA glycosylases: AAG1 that showed a robust activity against deamination lesions and AAG2 that was not active on the deamination lesions; nevertheless, the preferable substrate was identified as one of the exocyclic DNA adducts, 1,N6-ethenoadenine. Neither did TvAAG1 nor TvAAG2 compensate for the lack of OGG1 in T. vaginalis. In this chapter I also present studies of NTH1 from G. intestinalis. I have shown that GiNTH1 uses the same catalytic residues as its previously characterised homologs, the protein with deleted two extra amino acids within the iron-sulphur cluster retained majority of the activity. The wild type protein was found to possess a very weak ability to remove 8-oxoguanine from double-stranded oligonucleotide and even less activity was detected for the GiNTH1_Del. mutant. The preliminary experiments presented in this chapter provide the first evidence that human NTH1 iii containing a [2Fe-2S] iron-sulphur cluster not only is an active protein, but in particular, it is able to remove 8-oxoG from double-stranded oligonucleotide containing an 8-oxoG:C pair. The third chapter of results focuses on a polynucleotide 5´-kinase 3´-phosphatase (PNKP), an important enzyme of single-strand DNA break repair that typically works in response to oxidative damage within trypanosomatid parasites. PNKP is absent in microaerophilic organisms such as G. intestinalis or T. vaginalis, however curiously, PNKP was also found to be unexpectedly absent in certain obligately aerobic Leishmania parasites. I show that PNKP from T. brucei, appears to be the only one DNA repair enzyme with 5´-kinase activity and it contributes to the majority of 3’phosphatase activity. Here, I present that both PNKP -/- and PNKP -/-APE -/- cells are more sensitive to neocarzinostatin and camptothecin than the wild-type cells with only minor difference in cell viability between PNKP -/- and PNKP -/-APE -/- cell lines. In the fourth chapter of results, I demonstrat that apurinic/apyrimidinic endonuclease (APE1) from G. intestinalis possessed predicted AP endonuclease activity and additional phosphatase and non-specific exonuclease activities. Further site-directed mutagenesis studies demonstrated that mutation of aspartic acid at position 213 to alanine abolished the GiAPE1 activity. The phosphatase activity of GiAPE1 might compensate in G. intestinalis, at least to some extent, for the phosphatase activity of the absent PNKP protein.