Repair and adverse outcomes of damage in transcribed DNA
1 PhD project offered in the IPP summer call 2019
Deficient or aberrant transcriptional bypass of various types of damage in DNA (e.g., products of oxidative damage, adducts of environmental carcinogens or lesions generated by imbalanced repair of such damage) can lead to cytotoxic responses and changes of cell phenotype, thus emerging as a causal factor of degenerative diseases and accelerated ageing. This transcription-associated toxicity is counteracted by transcription-coupled nucleotide excision repair (TC-NER), a versatile repair pathway in which the damage recognition function is fulfilled by RNA polymerase. To get a better insight into the modes of interaction of DNA damage with transcription components in cells, our team has developed a procedure for targeted incorporation of synthetic nucleobase modifications into pre-defined positions in reporter vectors (Kitsera et al., 2011). Thus, we can combine chemical biology with genetic tools to dissect molecular steps of processing of structurally defined DNA modifications (DNA lesions or epigenetic marks) specifically positioned in the regulatory gene elements (Kitsera et al., 2017) or in transcribed DNA sequences (Kitsera et al., 2014; Lühnsdorf et al., 2014; Kitsera et al. 2016). This gives us possibility to identify critical repair intermediates and detect functional overlaps between the repair pathways for specific DNA lesions in human cells.
PhD project proposal: Co-transcriptional repair and transcriptional bypass of DNA damage
The scope of present project is straightforward characterisation of molecular events critical for nucleotide excision repair of transcribed genes. To underpin key molecular events of TC-NER we will induce stalling of elongating transcription complexes at the defined damage site in repair deficient cells and monitor their remodelling/displacement by mapping their components (Pol II, RNA:DNA hybrid) at the template DNA and characterising the associated proteins by quantitative mass spectrometry (in collaboration with Falk Butter lab). Next, we will address roles of the TC-NER pathway components (CSB, CSA, UVSSA, XAB2) in the recognition and processing of transcription complexes arrested at the damage site. Further (in collaboration with Thomas Carell lab), we will compare molecular responses to structurally different DNA lesions in transcribed DNA in order to identify determinants for their processing by the global genome (GG-) versus TC-NER pathways. In parallel, we will investigate the accuracy and efficiency of bypass of these lesions during RNA synthesis in cells. The results will contribute to understanding the links between the TC-NER defects and the associated degenerative diseases.
Kitsera N, Stathis D, Lühnsdorf B, Müller H, Carell T, Epe B, Khobta A (2011) 8-oxo-7,8-dihydroguanine in DNA does not constitute a barrier to transcription but is converted into transcription-blocking damage by OGG1. Nucleic Acids Res, 39, 5926-5934
Kitsera N, Allgayer J, Parsa E, Geier N, Rossa M, Carell T, Khobta A (2017) Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter Nucleic Acids Res, 45, 11033-11042
Kitsera N, Gasteiger K, Lühnsdorf B, Allgayer J, Epe B, Carell T, Khobta A (2014) Cockayne syndrome: varied requirement of transcription-coupled nucleotide excision repair for the removal of three structurally different adducts from transcribed DNA. PLoS One, 9, e94405
Lühnsdorf B, Epe B, Khobta A (2014) Excision of uracil from transcribed DNA negatively affects the gene expression. J Biol Chem, 289, 22008-22018
Allgayer J, Kitsera N, Bartelt S, Epe B, Khobta A (2016) Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine. Nucleic Acids Res, 44, 7267-7280