DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions... Show moreDNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol zeta) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity. Show less
Agrobacterium tumefaciens is a phytopathogen which is known to be the causal agent of crown gall disease in dicotyledonous plants. Virulent Agrobacterium strains are always carrier of a tumor... Show moreAgrobacterium tumefaciens is a phytopathogen which is known to be the causal agent of crown gall disease in dicotyledonous plants. Virulent Agrobacterium strains are always carrier of a tumor-inducing (Ti) plasmid. As a result of the expression of Vir proteins, a section of this plasmid termed the T-DNA is transferred via a type four secretion system (T4SS) as a T-strand to the recipient cell where it can integrate into the genomic DNA of the recipient cell. In this thesis the prerequisites of T-DNA circle formation using yeast as a model were assayed. It was found that the homologous repair (HR) protein Rad52 is of importance to the formation of T-DNA circles. In addition to this, the effects of double strand break (DSB) induction on the efficiency of T-DNA integration via the HR pathway in yeast and the role the nucleosome occupancy of the target locus has on DSB induction were assayed. It was found that a decrease in the nucleosome occupancy of a target locus had no measurable impact on the efficiency of T-DNA integration at this locus. However, the combined translocation of nuclease proteins aimed at the target locus and T-strands during AMT greatly facilitated HR-mediated integration of T-DNA. Show less
All organisms are composed of cells and the cell's nucleus contains DNA. The induction of DNA damage is a threat to organisms. Signalling of DNA damage and subsequent repair is of substantial... Show moreAll organisms are composed of cells and the cell's nucleus contains DNA. The induction of DNA damage is a threat to organisms. Signalling of DNA damage and subsequent repair is of substantial importance. Double-strand breaks (DSBs) in DNA can be induced by ionising radiation and DNA damaging agents but also arise as intermediates in several cellular processes (e.g. meiosis). DSBs are among the most genotoxic DNA lesions and their accurate repair is crucial. Genetic instability resulting from unrepaired DSBs can lead to cell death and, in a multicellular organism, to cancer. There are two major DSB repair pathways: homologous recombination (HR) and non-homologous endjoining (NHEJ). By NHEJ, broken DNA is sealed together, irrespective of sequence homology, in a not necessarily error-free way. HR, in which a homologous DNA molecule is needed as a template, accurately repairs DSBs. In this thesis we mainly focus on recombinational repair proteins of the RAD52 epistasis group and involved in HR. We analyse the biochemical properties of two Rad52 homologs in S. pombe, Rad22A and Rad22B. We examine combined mutations in RAD52 and RAD54 homologs in S. pombe and mice. We investigate the importance of sumolyation of recombinational repair proteins. We also introduce mice deficient in Sycp1, important for coordination during meiosis and the formation of crossovers Show less