Abstract

The integrity and stability of DNA is essential to life since it stores genetic information in every living cell. Chemicals from the environment will assault DNA to form various types of DNA damage, ranging from small covalent crosslinks between neighboring DNA bases as seen in cyclobutane pyrimidine dimers, to big bulky adducts derived from benzo[a]pyrene. This resultant damage will lead to replication block and mutation if remain unrepaired and will eventually cause cancer or other genetic diseases. The work presented in this dissertation has illustrated the important role of the AlkB family DNA repair enzymes in cancer and Wilson’s Disease. In addition, we discovered these enzymes can modify epigenetic markers that affect DNA regulation. We also studied sequence-dependent conformational heterogeneity of aminobiphenyl adduct on DNA replication. The AlkB family DNA repair enzyme is a family of α-ketoglutarate (αKG)- and non-heme iron-dependent dioxygenases. Among all the homologs in this family, human ALKBH2 and ALKBH3, and E. coli AlkB have been proved to be the major enzymes that directly remove the alkyl adducts from alkylated DNA bases like 3-methylcytosine (3mC) and 1-methyladenine (1mA). These DNA adducts will cause strong replication block and mutagenicity in cell if AlkB enzymes are suppressed by toxicants. Cancer-associated mutations often lead to perturbed cellular energy metabolism and accumulation of potentially harmful oncometabolites. Chiral molecule 2-hydroxyglutarate (2HG) and its two stereoisomers (D- and L-2HG) have been demonstrated to competitively inhibit several αKG- and iron-dependent dioxygenases, including ALKBH2 and ALKBH3. In this work, we carried out detailed kinetic analyses of DNA repair reactions catalyzed by ALKBH2, ALKBH3 and the bacterial AlkB in the presence of D- and L-2HG in both double and single stranded DNA contexts. We not only determined kinetic parameters of inhibition, including kcat, KM, and Ki, but also correlated the relative concentrations of 2HG and αKG previously measured in tumor cells with the inhibitory effect of 2HG on the AlkB family enzymes. Both D- and L-2HG significantly inhibited the human DNA repair enzymes ALKBH2 and ALKBH3 under pathologically relevant concentrations (73-88% for D-2HG and 31-58% for L-2HG inhibition). This work provides a new perspective that the elevation of either D- or L-2HG in cancer cells may contribute to an increased mutation rate by inhibiting the DNA repair carried out by the AlkB family enzymes and thus exacerbate the genesis and progression of tumors. Another type of inhibitor of AlkB is toxic metals, such as, copper. Disturbed

Highlights

  • The elevation of L-2HG under such conditions is key from either loss of expression of L-2HG dehydrogenase or promiscuous substrate utilization by lactate dehydrogenase A and malate dehydrogenases 1 and 2.10,12 Both D-2HG (R-2HG) and L-2HG (S-2HG) and several other molecules have been identified as oncometabolites because their accumulations in different tumor cells are originated from dysregulated energy metabolism pathways and metabolic imbalance

  • The ten-eleven translocation (TET) family enzymes belong to the α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases; they have been extensively studied in the last decade for their biological functions, biochemical activities and structural features.(1, 5, 6, 11) In the structures of the TET enzymes, the highly conserved N-terminal β-hairpin-like element for DNAbase recognition and the C-terminal catalytic domain are found in another family of α-KG/Fe(II)-dependent nucleic acid-modifying dioxygenases, the AlkB family proteins.(5, 12, 13) In this work, we show that the epigenetic modulator 5mC is modified in vitro to 5hmC, 5fC, and 5caC by the DNA repair enzymes in the AlkB family, including human ALKBH2, ALKBH3, and its Escherichia coli (E. coli) homolog AlkB

  • Different homologs of the E. coli AlkB protein exist in prokaryotic and eukaryotic species; nine homologs exist in human cells (ALKBH1-8 and FTO).(12, 14) Among the nine homologs, ALKBH2 and ALKBH3 are DNA repair enzymes that protect the informational integrity of the genome.(5, 14–18) They use an α-KG/Fe(II)-dependent mechanism to oxidize aberrant alkyl groups, restoring the undamaged DNA bases.(14, 17, 18) The reported substrate scope of AlkB, ALKBH2 and ALKBH3 includes 3-methylcytosine (3mC, Figure 1), 1-methyladenine (1mA), 3-methylthymine (3mT) and 1-methylguanine (1mG), as well as other nitrogen-attached methyl lesions occurring at the Watson-Crick base pairing interface of DNA bases.(15, 16, 18–20)

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Summary

Introduction

Mutations in the isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) are frequently found in >75% human low grade glioma, secondary glioblastoma, cartilaginous tumor and >20% of acute myeloid leukemia. Tumor-derived mutant forms of IDH catalyze the NAD-dependent dehydrogenation of α-ketoglutarate (αKG) to D-2hydroxyglutarate (D-2HG), a function that supplants the physiological activity of IDH, which entails reductive decarboxylation of isocitrate to αKG (Figure 1b). L-2HG, the stereoisomer of D-2HG, has been identified as an oncometabolite with elevated concentrations in renal cell carcinoma neurodegenerative disorders, and in tissues under oxygen limitation or hypoxic conditions. The elevation of L-2HG under such conditions is key from either loss of expression of L-2HG dehydrogenase or promiscuous substrate utilization by lactate dehydrogenase A and malate dehydrogenases 1 and 2.10,12 Both D-2HG (R-2HG) and L-2HG (S-2HG) and several other molecules have been identified as oncometabolites because their accumulations in different tumor cells are originated from dysregulated energy metabolism pathways and metabolic imbalance. Because of their structural similarity to αKG, both D- and L2HG could compete with αKG and inhibit enzymatic processes that use αKG as a substrate. A careful study of the inhibitory effect of both D- and L-2HG on AlkB repair enzymes is needed to quantify the extent of inhibition of the direct reversal DNA repair pathways; perturbations in these pathways would lead to unrepaired mutagenic DNA lesions, which would cause mutations that can accelerate tumor progression or enable metastatic growth Such a study would facilitate the identification of druggable targets related to the AlkB enzymes because many alkylating chemotherapeutic agents generate DNA adducts that are repaired by this family of repair enzymes.. These bulky-lesion induced conformational heterogeneities complicate mutational and repair outcomes [9-11, 1315]

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