Molecular architecture and function of adenovirus DNA polymerase

Central to this thesis is the role of adenovirus DNA polymerase (Ad pol) in adenovirus DNA replication. Ad pol is a member of the family B DNA polymerases but belongs to a distinct subclass of polymerases that use a protein as primer. As Ad pol catalyses both the initiation and elongation phases and needs to accomodate both DNA and protein as a primer, it is not surprising that a large number of protein-protein and protein-DNA interactions are involved in efficient replication. Indeed, Ad pol is known to interact with pTP, NFI and DNA, although our understanding of these interactions is limited. In this thesis, these interactions have been studied in greater detail. After an introductory chapter on DNA dependent DNA polymerases and Ad replication, the jumping back mechanism that characterizes the change from initiation to elongation is extensively reviewed in chapter 2. In chapter 3, the highly conserved (I/Y)XGG motif of Ad pol is studied. In chapter 4, the interaction between Ad pol and DNA is further studied by the use of biotinylated oligo-nucleotides with a bulky streptavidin block. Chapter 5 examines the termination of Ad pol on the native TP-containing viral DNA. Finally, in chapter 6 the recruitment of the pTP-pol complex via a direct interaction between Ad pol and NFI is studied in detail

SIRT1 mediates FOXA2 breakdown by deacetylation in a nutrient-dependent manner

The Forkhead transcription factor FOXA2 plays a fundamental role in controlling metabolic homeostasis in the liver during fasting. The precise molecular regulation of FOXA2 in response to nutrients is not fully understood. Here, we studied whether FOXA2 could be controlled at a post-translational level by acetylation. By means of LC-MS/MS analyses, we identified five acetylated residues in FOXA2. Sirtuin family member SIRT1 was found to interact with and deacetylate FOXA2, the latter process being dependent on the NAD +-binding catalytic site of SIRT1. Deacetylation by SIRT1 reduced protein stability of FOXA2 by targeting it towards proteasomal degradation, and inhibited transcription from the FOXA2-driven G6pase and CPT1a promoters. While mutation of the five identified acetylated residues weakly affected protein acetylation and stability, mutation of at least seven additional lysine residues was required to abolish acetylation and reduce protein levels of FOXA2. The importance of acetylation of FOXA2 became apparent upon changes in nutrient levels. The interaction of FOXA2 and SIRT1 was strongly reduced upon nutrient withdrawal in cell culture, while enhanced Foxa2 acetylation levels were observed in murine liver in vivo after starvation for 36 hours. Collectively, this study demonstrates that SIRT1 controls the acetylation level of FOXA2 in a nutrient-dependent manner and in times of nutrient shortage the interaction between SIRT1 and FOXA2 is reduced. As a result, FOXA2 is protected from degradation by enhanced acetylation, hence enabling the FOXA2 transcriptional program to be executed to maintain metabolic homeostasis

The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response

In this study, we searched for proteins regulating the tumor suppressor and life-span regulator FOXO4. Through an unbiased tandem-affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double-strand break repair component. Using biochemical interaction studies, we found Ku70 to be necessary and sufficient for the interaction. FOXO4 mediates its tumor-suppressive function in part through transcriptional regulation of the cell cycle arrest p27kip1gene. Immunoblotting, luciferase reporter assays, and flow cytometry showed that Ku70 inhibited FOXO4-mediated p27kip1transcription and cell cycle arrest induction by >40%. In contrast, Ku70 RNAi but not control RNAi significantly increased p27kip1transcription. In addition, in contrast to wild-type mouse embryonic stem (ES) cells, Ku70-/-ES cells showed significantly increased FOXO activity, which was rescued by Ku70 reexpression. Immunofluorescence studies demonstrated that Ku70 sequestered FOXO4 in the nucleus. Interestingly, the Ku70-FOXO4 interaction stoichiometry followed a nonlinear dose-response curve by hydrogen peroxide-generated oxidative stress. Low levels of oxidative stress increased interaction stoichiometry up to 75%, peaking at 50 μM, after which dissociation occurred. Because the Ku70 ortholog in the roundworm Caenorhabditis elegans was shown to regulate life span involving C. elegans FOXO, our findings suggest a conserved critical Ku70 role for FOXO function toward coordination of a survival program, regulated by the magnitude of oxidative damage