Krawczyk, Claudia. Novel functional aspects of topoisomerase Top1 and DNA Glycosylase Thp1 in the maintenance of genetic and epigenetic stability in Yeast. 2014, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_11108
DNA replication forks (RFs) are frequently blocked by different obstacles, such as programmed RF barriers (RFBs) found in many organisms. The well-studied ribosomal RFB (rRFB) of budding yeast requires binding of Fob1 for its activity, a protein that is important for the stability of the highly repetitive ribosomal DNA (rDNA). Fob1 also mediates the reported anchoring of the rDNA repeats to the inner nuclear membrane. The resulting restricted structural flexibility creates a great amount of torsional stress, which is normally removed by DNA topoisomerases that cut the DNA by forming a covalent intermediate to allow for DNA relaxation before re-ligation. Indeed, topoisomerases are important for rDNA stability and topoisomerase 1 (Top1)-dependent nicks were mapped close to the rRFB. As it remained elusive how Top1 action is regulated in the rDNA, the first aim of my thesis was the identification of factors influencing Top1 nicking activity within the rDNA and particularly at the rRFB in Saccharomyces cerevisiae.
I found that Top1-DNA complexes (Top1 cleavage complexes, Top1ccs) and, thus, DNA nicks are stabilized at the rRFB. Top1cc accumulation requires Fob1 and the nucleolar protein Tof2, but not RF stalling. Interestingly, Top1cc stabilization by Fob1 and Tof2 does not require the rDNA context, as Fob1-dependent Top1ccs also accumulated at an ectopically located rRFB that was neither recruited to the nucleolus nor to the inner nuclear membrane. We also identified Top1 nicks to account for most of the DNA double-strand breaks (DSBs) previously described to arise in wild-type cells, pointing at an in vitro conversion of single-stranded Top1 nicks to DSBs. On the basis of these data, we propose a model in which Top1 is recruited to the rRFB, where it nicks the DNA forming a stable Top1cc intermediate to allow for continuous relaxation, thereby contributing to the genetic stabilization of the structurally complex locus. This stabilization would be achieved by protein-protein interactions with Fob1 and Tof2 or by misalignment of the break end by the structure of the complex. In the published manuscript we thus present first evidence for locus-specific regulation of Top1 catalytic actions.
Besides regulating DNA torsion and ensuring RF integrity, genome maintenance also involves the removal of irregular DNA bases. Uracil deriving from cytosine deamination or uracil misincorporation during DNA replication is excised by uracil DNA glycosylases (UDGs) to initiate base excision repair (BER) that restores the correct base pair. In mammals, four nuclear UDGs with partially overlapping functions are expressed. While UNG2 and SMUG1 were mainly associated with “classical” uracil excision repair, TDG and MBD4 appear to be important for epigenetic regulation of gene expression. The functional separation of the single enzymes, however, is not fully understood. Thus, the second aim of my thesis was to further separate UDG functions by using the less complex Schizosaccharomyces pombe model that expresses only the UNG2 ortholog Ung1 and the TDG ortholog Thp1, and that has chromatin regulation mechanisms similar to mammalian cells, while having no DNA methylation.
In genetic studies, we found that despite a dominant uracil excision activity of Ung1 in cell-free extracts, both Ung1 and Thp1 contribute to uracil removal and mutation avoidance in vivo. Interestingly, Thp1 expression mediates cytotoxicity during 5-fluorouracil (5-FU) exposure and in the presence of increased amounts of genomic uracil, most likely reflecting the reported high affinity of Thp1 to its product abasic site (AP-site). Presence of AP sites could occasionally lead to DNA breakage and, indeed, most spontaneous mitotic recombination events depend on Thp1 but not on Ung1. Thus, Ung1 and Thp1 have overlapping functions in uracil removal, however, Ung1-mediated BER appears fast and mostly error-free while Thp1-dependent BER seems slow and error-prone. Given the role of mammalian TDG in regulating gene expression, we compared genome-wide RNA expression in Thp1-proficient and -deficient strains and observed a mild but overall down-regulation and a hyper-variability of gene expression in the absence of Thp1. Thus, Thp1 might contribute to the maintenance of transcriptionally active chromatin and S. pombe may serve as a model for studying the impact of UDGs on gene expression.
Taken together, my work provided insights into a novel regulatory aspect of Top1 function in the rDNA of budding yeast and into the function of Ung1- and Thp1-dependent uracil repair in S. pombe.
|Committee Members:||Boiteux, Serge|
|Faculties and Departments:||03 Faculty of Medicine > Departement Biomedizin > Division of Biochemistry and Genetics > Molecular Genetics (Schär)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||1 Bd.|
|Last Modified:||30 Jun 2016 10:56|
|Deposited On:||02 Feb 2015 13:24|
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