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Clausing, Emanuel (2008): Characterization of the Cyclin Dependent Kinase Complex Bur1-2 and its Interaction with RPA. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Gene expression is highly regulated and interconnected to processes like mRNP processing, mRNA export as well as to DNA repair and replication. The first step of gene expression is the transcription of protein coding genes by RNA polymerase II. Transcription is controlled by general transcription factors, the phosphorylation of the C-terminal domain of Rpb1, the largest subunit of RNA polymerase II, and chromatin modifications that allow proper accessibility of the DNA. A major player in these coupling processes is the TREX complex, coupling transcription elongation to the nucleo-cytoplasmic export of the mRNP via the nuclear pore complex. Particularly, the THO subcomplex of TREX has functions in hyperrecombination, nucleotide excision repair and transcription coupled repair. A genetic screen with TREX components, performed to identify genes involved in these processes, lead to the identification of the cyclin dependent kinase Bur1. Bur1 and its cyclin Bur2 are needed for efficient transcription elongation by RNA polymerase II by regulating the methylation of histone tails. Interestingly, Bur1 interacts in vivo with RPA, a single strand DNA binding protein essential for genome stability. This biochemical interaction raised the idea of a novel interconnection between transcription, chromatin modification and genome maintenance. Mutations in the BUR1 as well as in the RFA1 gene lead to sensitivity to drugs that cause DNA damage and replication or transcription stress. Deletion of the C-terminus of Bur1, which is sufficient for the binding to RPA, also renders cells sensitive to those agents. This shows the functional significance of this protein-protein interaction in the cell upon stress induction. However, attempts to identify the DNA repair pathway Bur1 is involved in showed that mutations in BUR1 do not behave epistatic with deletions of specific pathways. This result points to a more general, maybe regulatory role of Bur1 in the response to DNA damage. It is interesting to note that mutations in BUR1 lead to increased genomic instability as they show the appearance of a higher amount and longer persistence of nuclear foci, DNA repair “factories” that contain, among other proteins, Rfa1 and Rad52. Furthermore, RFA1 mutants show decreased levels of histone H3 alone as well as lower levels of histone H3 Lysine 4 trimethylation, a mark for transcription elongation, when combined with a mutation in BUR1. The RFA1 mutant is also impaired in the expression of a β-galactosidase reporter gene, pointing to a function of RPA in transcription. Interestingly, combining BUR1 and RFA1 mutants leads to a lower susceptibility of cells to stress than one of the mutations alone. On the one hand, this could be elucidated by better growth of the double mutant strains upon stress compared to the single mutants. On the other hand, whole genome expression analysis shows that the double mutant strain clusters with the bur1 mutant whereas the rfa1 mutant does not, showing that its expression pattern is closer to the bur1 mutant. Both results show that the protein complexes have antagonistic roles as the combination of both mutations leads to a suppression phenotype based on differential gene expression. Taken together, a function of Bur1 in genome maintenance could be established, as well as an effect of RPA on transcription elongation and chromatin modification. The results provide a possibility to speculate about a coupling of transcription and genome stability mediated by the interaction of Bur1-2 with RPA.