Dynamics of the human nuclear proteome in response to DNA damage

Abstract

The genome is constantly challenged by factors that can induce DNA damage and thereby threaten the viability of the cell. If DNA damage remains unrepaired it can lead to the development of cancer. Although much is known about the role of proteins and protein complexes in the cellular response to DNA damage, an overview of events occurring at the protein level upon DSB induction has never been reported. Here proteomics was used to bring this about. Mapping of cellular events at the proteome level requires methods that allow accurate and reproducible quantitation of protein expression levels in complex biological systems. We compared a 2D gel-based quantitation method, two-dimensional difference in-gel electrophoresis (2D-DiGE), to metabolic stable isotope labelling, one of the most accurate methods. This showed that both methods provide comparable results with excellent correlation. Subsequently, 2D-DiGE was applied to the investigation of effects of bleomycin-induced DNA damage on the nuclear proteome of human lymphoblastoid cell lines. Interestingly, the nuclear levels of three proteins, known to form the inhibitor of acetyltransferase (INHAT) complex involved in transcriptional regulation, were found to decrease rapidly upon DNA DSB induction, suggesting a role for the INHAT complex in chromatin remodelling events that precede DNA repair. Some of the cell lines used in our study show a hypersensitive phenotype, which means they are hypersensitive towards DNA damage induction and the individuals from which they were retrieved have an increased risk to develop cancer. Since the underlying mechanisms for this decreased genome stability are unknown, we attempted to gain insight into the way these cells cope with DNA damage. This revealed differences in nuclear protein levels between the normal and the hypersensitive cell lines and suggested that differences in regulation of chromatin structure can also influence cellular sensitivity towards DNA damage induction. In the tight regulation of the response to DNA damage, protein phosphorylation plays a crucial role. To study this, a method was developed for the specific enrichment of serine- and threonine-phosphorylated peptides, which is based on the base-catalysed b-elimination of phosphate from a serine- or threonine-phosphorylated peptide. The method allowed selective enrichment of a phosphorylated peptide from a peptide mixture. Additionally, DNA damage-induced changes in the sub-proteome of histone-binding proteins were studied. Despite the growing interest in the role of chromatin remodelling in the DNA damage response, not much is known about the exact mechanisms through which these processes are regulated. Our study reveals that phosphorylation plays an important role in activation of histone chaperones that take part in multiple remodelling complexes. Our results provide insight into some complex features of protein networks at the cross roads of nucleosome assembly, DNA transcription and repair. Taken together, the work described in this thesis highlights the strength of proteomics when analysing complex cellular mechanisms, like the DNA damage response and it emphasizes the importance of protein post-translational modifications and relocalization in the regulation of cellular events.