Repair of genomic uracil and ribonucleotides - Regulation and potential involvement in new and old cancer treatments

Abstract

Life as we know it has evolved over millions of years and evolution has produced complex biochemical machineries that exist in every living cell. Many of these processes may appear imperfect and to threaten the stability of genetic material. However, research shows that what may seem imperfect may be essential for cell survival and can represent important drug targets, especially in cancer treatment. All cells devote significant amounts of energy to produce proteins that maintain its genetic material. In this thesis, we primarily investigated Uracil-DNA glycosylase (UNG). The protein exists in the nucleus as UNG2, and in the mitochondria as UNG1, where it recognizes and removes the base uracil (U) from DNA before further repair processes restores the code. Due to evolutionary processes, hundreds of thousands of uracil (U) bases are incorporated instead of the building block thymine (T) during copying of DNA (replication). Uracil also arises from deamination of the normal building block cytosine ©. This occurs spontaneously due to the instability of cytosine but may also happen enzymatically. APOBEC proteins, which are part of the cell’s viral defense, and AID, which participates in antibody production, target C for deamination to U. Collateral damage caused by AID/APOBEC that is left unrepaired has been linked to the development of over half of all human cancers! In paper I we discovered that a new class of chemotherapy used against B- and T-cell lymphomas, histone deacetylase inhibitors (HDACi), surprisingly led to specific degradation of UNG2. HDACi also reduced the synthesis of T for DNA replication and contributed to increased U levels in the genome. Proteome wide studies revealed several previously undescribed protein changes that help understanding HDACi’s effects on cancer and disease. In paper II we used a novel assay that showed that UNG1/2 could remove uracil from singlestranded DNA, even when protected by the single-stranded binding protein RPA. The activity depended on specific interaction domains in RPA and UNG1/2 that is regulated by amino acid modifications in the surrounding area. Our findings have improved the understanding of how genomic uracil is processed throughout the cell cycle, especially during DNA replication, and how this impacts antibody formation in immune cells. In paper III, we examined whether HDACi could enhance the effect of pemetrexed (PMX), an antifolate that inhibits the production of the DNA building block T. It has been thought PMX leads to increased incorporation of U during DNA copying and the risk of large DNA damage, such as double-strand breaks, after repeated U incorporation and futile repair cycles after UNG initiated repair. The combination of PMX with HDACi resulted in strong activation of DNA damage markers, but this was not caused by UNG2 degradation, as cancer cell lines without UNG2 showed no difference in sensitivity or activation of DNA damage markers following PMX treatment. To map alternative cytotoxic mechanisms of PMX and HDACi, we conducted large-scale proteomic analyses following PMX and/or HDACi treatment. Based on our findings, we propose a new model for the cytotoxicity of antifolates like PMX, suggesting it is caused by the increased misincorporation of RNA building blocks into DNA. These are mainly repaired by a process like uracil repair but initiated by the enzyme RNaseH2, which is also degraded by HDACi. RNA building blocks are inherently toxic when incorporated in DNA and may enhance the effects of immunotherapy, however, alternative repair pathways are particularly cytotoxic. This model is important to explore further and may contribute to understanding the mechanisms of action of several common chemotherapies. Throughout this work, we have described new mechanisms of function and regulation of UNG1/2 and revealed that both new and old cancer drugs affect UNG-related processes. UNG1 and UNG2 have served as model proteins for several DNA repair proteins for many years, and the knowledge gained can be applied to other proteins. This work also provides valuable insight into widely used drugs. Still, we are far away from completely understanding the complex cell machinery that is the basis for disease and life itself.