Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Deyu Li

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 metabolism of copper ions can cause diseases, such as Wilson’s disease (WD). In this work, we investigated the inhibitory effect of Cu(II) ion on the AlkB family DNA repair enzymes, include human ALKBH2, ALKBH3, and E. coli AlkB proteins. None of the three proteins were significantly inhibited under normal cellular copper concentrations. But under WD related condition, we observed the activities of all three enzymes were strongly suppressed (inhibition from 95.2 to 100.0%). We also noted the repair efficiency under ds-DNA condition is less susceptible than ss-DNA to the inhibition.

AlkB can repair many alkylated DNA bases including 3mC, 1mA, 3-metheylthymine, 1-methylguanine, ethenoadenine and ethenocytosine. But in this work, we found a new DNA base substrate for AlkB, 5-Methylcytosine (5mC). 5mC in DNA CpG islands is an important epigenetic biomarker for mammalian gene regulation. It is oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the ten-eleven translocation (TET) family enzymes, which are also α-KG/Fe(II)-dependent dioxygenases. In this work, we demonstrate that the epigenetic marker 5mC is biochemically modified to 5hmC, 5fC, and 5caC by ALKBH2, ALKBH3, and AlkB. Theoretical calculations indicate that these enzymes may bind 5mC in the syn-conformation, placing the methyl group comparable to 3-methylcytosine, the prototypic substrate of AlkB. This is the first demonstration of the AlkB proteins to oxidize a methyl group attached to carbon, instead of nitrogen, on a DNA base. These observations suggest a broader role in epigenetics for these DNA repair proteins.

Besides alkyl DNA adducts, there are bulky DNA adducts existing in human. Bulky organic carcinogens are activated in vivo and subsequently react with nucleobases of cellular DNA to produce adducts. Some of these DNA adducts exist in multiple conformations that are slowly interconverted to one another. Different conformations could contribute to different mutagenic and repair outcomes. Unfortunately, studies on the conformation-specific inhibition of replication, which is more relevant to cell survival, are scarce; this is presumably due to difficulties in studying the structural dynamics of DNA lesions at the replication fork. It is challenging to capture the exact nature of replication inhibition by traditional end-point assays, since they usually detect either the ensemble of consequences of all the conformers or the culmination of all cellular behaviors, such as mutagenicity or survival rate. One article reported an unusual sequence-dependent conformational heterogeneity involving FABP-modified (4′-fluoro-4-aminobiphenyl) DNA under different sequence contexts. There were 67% B-type (B) conformation and 33% stacked (S) conformation in TG1*G2T sequence; whereas, 100% B conformation was observed in TG1G2*T sequence. In this study, we applied primer extension assay to compare the inhibition models between these two FABP-modified DNA sequences. We utilized a combination of surface plasmon resonance (SPR) and HPLC-based steady-state kinetics to reveal the differences in terms of binding affinity and inhibition with polymerase between these two conformers (67%B:33%S and 100%B). The conformational heterogeneities from these two sequences lead to different types of inhibition on replication.

Available for download on Thursday, October 17, 2019

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