The Interactomics of DNA Repair and Genome Maintenance

Proteins are the building blocks as well as the construction workers of the cells. Many proteins are enzymes that catalyze the thousands of biochemical reactions in each cell. It has gradually become evident that many individual proteins can have functions in different cellular pathways. This can be facilitated by a protein having intrinsically disordered regions (IDRs) that can allow modulated binding surfaces to different protein partners or to other macromolecules such as DNA/RNA.The molecular mechanisms underlying switching from one partner to another are yet poorly characterized, but often encompass post-translational modifications such as phosphorylations, acetylations or ubiquitinylations in or closely spaced relative to IDR. We have for nearly three decades studied the enzyme Uracil-DNA glycosylase (UNG2), which removes the anomalous nitrogen base uracil from DNA and thus hinders mutations. Uracil is often misincorporated during DNA replication, and the location of UNG2 at the replication fork by binding to the replication proteins PCNA and RPA ensures efficient removal of misincorporated uracil. UNG2 can, however also bind to other proteins, like the HIV-1-encoded protein Vpr, and is also crucial for correct maturation of antibodies. We have previosly determined the 3-dimensional structure of the compact catalytic domain of UNG2 co-crystallized with uracil-containing DNA, and have shown at the binding induces "flipping" of uracil out of the helix and into the active site of the enzyme to cleave uracil from the deoxyribose. In addition, UNG2 possesses a partially unstructured N-terminal region of approximately 85 amino acids, that contain the PCNA- and RPA-binding motifs and also apparently fine-tunes the enzymatic activity. Likely, this region also contain hitherto unknown binding elements. We have until now achieved NMR-assignments of the folded and the intrinsic disordered N-terminal domain in UNG2. This constitutes a map of all protein nuclei and allows extraction of information about binding surfaces and conformational changes at different experimental conditions. One unexpected finding of this was that the anticipated disordered region in UNG2 containes a long semistable alpha-helix mediates binding to RPA. The binding region is larger than previously postulated, and the binding about 10X stronger. We will now undertake simulations with our partners at CNRS-IBS in Grenoble. We have also started a collaboration with an NMR group in Copenhagen to study the interaction between UNG2 and PCNA. PCNA interacts with UNG2 via a conserved (PIP) motif, which places UNG2 at an ideal position to rapidly excise misincorporated Uracil. A central question here is how UNG2 can relocalize from PCNA to RPA, which binds single-stranded DNA in front of the replication fork. Such single-stranded regions rapidly expands when DNA-replication is blocked, and are subject to increased rates of cytosine deamination. Uracil originating from deaminated cytosine is 100% mutagenic if not repaired before entering the replication machinery. We aim at alalyzing conformational alterations occurring in the entire N-terminal by binding to RPA/PCNA, and whether the two motifs reciprocally may affect each others structures. Finally, we have affinity-enriched UNG2-containing protein complexes from B-cells prior to- and after stimulation to undergo class switch recombination. These comblexes will be analyzed by mass spectrometry to identify novel binders, and also potential mediation of binding by post-translational modifications. we have conducted a similar study by capturing proteins binding to activation-induced deaminase (AID), which acts upstream of UNG2 during antibody maturation. Identification of potentially common proteins in the complexes might reveal how the process as such is structurally orchestrated.

The single most important factor for the correct functioning of genomes is that their coding information is maintained intact and transferred unperturbed to future generations. DNA repair is highly coordinated- and integrated with other fundamental cellul ar processes such as transcription, replication, cell-cycle control and apoptosis. Core elements of DNA repair has even been adapted as essential parts of the adaptive immune system in higher vertebrates. Thus, a fundamental understanding of the molecular mechanisms underlying genomic maintenance is central to our understanding of regulation of genomic function. The present project was initiated as a FUGE II-project in 2007, and has resulted in ground-breaking knowledge about the fine-tuning of DNA repai r itself and cooperation with other cellular processes. An emerging picture is that complex post-translational modifications, often in intrinsically disordered regions of DNA repair proteins, dictate assembly of downstream protein complexes with distinct functions at the DNA interface. Here we focus on processing of genomic uracil as a model system to study these mechanisms at the molecular level. This is substantiated by or findings that core proteins in genomic uracil-initiation (AID) and processing (U NG2) apparently constitute central switch-points in repair versus mutagenic uracil processing. At the cellular level this regulation is crucial, and several lines of evidence indicate that dysregulation of the balance between error-fre and mutagenic uraci l-processing is intimately linked to cancer, especially B-cell cancers. Our long-standing experience within genomic uracil processing, the accumulated expertise in protein PTM analysis at the Proteomics and Metabolomics Core Facility (PROMEC), our close c ollaboration with the K.G. Jebsen Center for Myeloma Research at IKM as well as our outstanding international collaborators, should contribute to a high likelihood of success within the project.