CellTool: An Open-Source Software Combining Bio-Image Analysis and Mathematical Modeling for the Study of DNA Repair Dynamics.

Elucidating the dynamics of DNA repair proteins is essential to understanding the mechanisms that preserve genomic stability and prevent carcinogenesis. However, the measurement and modeling of protein dynamics at DNA lesions via currently available image analysis tools is cumbersome. Therefore, we developed CellTool-a stand-alone open-source software with a graphical user interface for the analysis of time-lapse microscopy images. It combines data management, image processing, mathematical modeling, and graphical presentation of data in a single package. Multiple image filters, segmentation, and particle tracking algorithms, combined with direct visualization of the obtained results, make CellTool an ideal application for the comprehensive analysis of DNA repair protein dynamics. This software enables the fitting of obtained kinetic data to predefined or custom mathematical models. Importantly, CellTool provides a platform for easy implementation of custom image analysis packages written in a variety of programing languages. Using CellTool, we demonstrate that the ALKB homolog 2 (ALKBH2) demethylase is excluded from DNA damage sites despite recruitment of its putative interaction partner proliferating cell nuclear antigen (PCNA). Further, CellTool facilitates the straightforward fluorescence recovery after photobleaching (FRAP) analysis of BRCA1 associated RING domain 1 (BARD1) exchange at complex DNA lesions. In summary, the software presented herein enables the time-efficient analysis of a wide range of time-lapse microscopy experiments through a user-friendly interface.

Interactions between HIV protease inhibitor ritonavir and human DNA repair enzyme ALKBH2: a molecular dynamics simulation study.

The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA. Overexpression of ALKBH2 has been implicated in both tumorigenesis and chemotherapy resistance in some cancers, including glioblastoma and renal cancer rendering it a potential therapeutic target and a diagnostic marker. However, no inhibitor is available against these important DNA repair proteins. Intending to repurpose a drug as an inhibitor of ALKBH2, we performed in silico evaluation of HIV protease inhibitors and identified Ritonavir as an ALKBH2-interacting molecule. Using molecular dynamics simulation, we elucidated the molecular details of Ritonavir-ALKBH2 interaction. The present work highlights that Ritonavir might be used to target the ALKBH2-mediated DNA alkylation repair.

Conformational Dynamics of Human ALKBH2 Dioxygenase in the Course of DNA Repair as Revealed by Stopped-Flow Fluorescence Spectroscopy.

Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of Escherichia coli AlkB dioxygenase. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O2. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. The results shed light on overall conformational dynamics of ALKBH2 and damaged DNA during the catalytic cycle.

MV1035 Overcomes Temozolomide Resistance in Patient-Derived Glioblastoma Stem Cell Lines.

Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain tumor and patients with GBM have a poor prognosis. Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represents the standard strategy for GBM. We showed that the imidazobenzoxazin-5-thione MV1035 is able to significantly reduce GBM U87-MG cells migration and invasiveness through inhibition of the RNA demethylase ALKBH5. In this work, we focus on the DNA repair protein ALKBH2, a further MV1035 target resulting from SPILLO-PBSS proteome-wide scale in silico analysis. Our data demonstrate that MV1035 inhibits the activity of ALKBH2, known to be involved in GBM TMZ resistance. MV1035 was used on both U87-MG and two patient-derived (PD) glioma stem cells (GSCs): in combination with TMZ, it has a significant synergistic effect in reducing cell viability and sphere formation. Moreover, MV1035 induces a reduction in MGMT expression in PD-GSCs cell lines most likely through a mechanism that acts on MGMT promoter methylation. Taken together our data show that MV1035 could act as an inhibitor potentially helpful to overcome TMZ resistance and able to reduce GBM migration and invasiveness.

Sequence Dependent Repair of 1,N6-Ethenoadenine by DNA Repair Enzymes ALKBH2, ALKBH3, and AlkB.

Mutation patterns of DNA adducts, such as mutational spectra and signatures, are useful tools for diagnostic and prognostic purposes. Mutational spectra of carcinogens derive from three sources: adduct formation, replication bypass, and repair. Here, we consider the repair aspect of 1,N6-ethenoadenine (εA) by the 2-oxoglutarate/Fe(II)-dependent AlkB family enzymes. Specifically, we investigated εA repair across 16 possible sequence contexts (5'/3' flanking base to εA varied as G/A/T/C). The results revealed that repair efficiency is altered according to sequence, enzyme, and strand context (ss- versus ds-DNA). The methods can be used to study other aspects of mutational spectra or other pathways of repair.

Boosting 5-ALA-based photodynamic therapy by a liposomal nanomedicine through intracellular iron ion regulation.

5-Aminolevulinic acid (5-ALA) has been approved for clinical photodynamic therapy (PDT) due to its negligible photosensitive toxicity. However, the curative effect of 5-ALA is restricted by intracellular biotransformation inactivation of 5-ALA and potential DNA repair of tumor cells. Inspired by the crucial function of iron ions in 5-ALA transformation and DNA repair, a liposomal nanomedicine (MFLs@5-ALA/DFO) with intracellular iron ion regulation property was developed for boosting the PDT of 5-ALA, which was prepared by co-encapsulating 5-ALA and DFO (deferoxamine, a special iron chelator) into the membrane fusion liposomes (MFLs). MFLs@5-ALA/DFO showed an improved pharmaceutical behavior and rapidly fused with tumor cell membrane for 5-ALA and DFO co-delivery. MFLs@5-ALA/DFO could efficiently reduce iron ion, thus blocking the biotransformation of photosensitive protoporphyrin IX (PpIX) to heme, realizing significant accumulation of photosensitivity. Meanwhile, the activity of DNA repair enzyme was also inhibited with the reduction of iron ion, resulting in the aggravated DNA damage in tumor cells. Our findings showed MFLs@5-ALA/DFO had potential to be applied for enhanced PDT of 5-ALA.

Oxidative demethylase ALKBH5 repairs DNA alkylation damage and protects against alkylation-induced toxicity.

DNA integrity is challenged by both exogenous and endogenous alkylating agents. DNA repair proteins such as Escherichia coli AlkB family of enzymes can repair 1-methyladenine and 3-methylcytosine adducts by oxidative demethylation. Human AlkB homologue 5 (ALKBH5) is RNA N6-methyladenine demethylase and not known to be involved in DNA repair. Herein we show that ALKBH5 also has weak DNA repair activity and it can demethylate DNA 3-methylcytosine. The mutation of the amino acid residues involved in demethylation also abolishes the DNA repair activity of ALKBH5. Overexpression of ALKBH5 decreases the 3-methylcytosine level in genomic DNA and reduces the cytotoxic effects of the DNA damaging alkylating agent methyl methanesulfonate. Thus, demethylation by ALKBH5 might play a supporting role in maintaining genome integrity.

Boosting 5-ALA-based photodynamic therapy by a liposomal nanomedicine through intracellular iron ion regulation

5-Aminolevulinic acid (5-ALA) has been approved for clinical photodynamic therapy (PDT) due to its negligible photosensitive toxicity. However, the curative effect of 5-ALA is restricted by intracellular biotransformation inactivation of 5-ALA and potential DNA repair of tumor cells. Inspired by the crucial function of iron ions in 5-ALA transformation and DNA repair, a liposomal nanomedicine (MFLs@5-ALA/DFO) with intracellular iron ion regulation property was developed for boosting the PDT of 5-ALA, which was prepared by co-encapsulating 5-ALA and DFO (deferoxamine, a special iron chelator) into the membrane fusion liposomes (MFLs). MFLs@5-ALA/DFO showed an improved pharmaceutical behavior and rapidly fused with tumor cell membrane for 5-ALA and DFO co-delivery. MFLs@5-ALA/DFO could efficiently reduce iron ion, thus blocking the biotransformation of photosensitive protoporphyrin IX (PpIX) to heme, realizing significant accumulation of photosensitivity. Meanwhile, the activity of DNA repair enzyme was also inhibited with the reduction of iron ion, resulting in the aggravated DNA damage in tumor cells. Our findings showed MFLs@5-ALA/DFO had potential to be applied for enhanced PDT of 5-ALA.

Non-heme dioxygenases in tumor hypoxia: They're all bound with the same fate.

Tumor tissues are known to harbor hypoxic areas. The hypoxic microenvironment promotes angiogenesis. Hypoxic tumor cells also manifest genome instability. DNA damage repair pathways, such as double-strand break repair, mismatch repair and base excision repair are known to be altered during hypoxia. This review is focused on the non-heme Fe(II) and 2-oxoglutarate-dependent dioxygenases which are involved in repair of DNA alkylation adducts. Activities of these DNA repair enzymes are completely oxygen-dependent and little information is available about inhibition of these enzymes during hypoxia. While impairment of function of non-heme dioxygenase during tumor hypoxia has been implicated in different studies, the possible outcomes with respect to mutagenesis and genomic instability are explored here.

The interaction between ALKBH2 DNA repair enzyme and PCNA is direct, mediated by the hydrophobic pocket of PCNA and perturbed in naturally-occurring ALKBH2 variants.

Human AlkB homolog 2 (ALKBH2) is a DNA repair enzyme that catalyzes the direct reversal of DNA methylation damage through oxidative demethylation. While ALKBH2 colocalizes with proliferating cell nuclear antigen (PCNA) in DNA replication foci, it remains unknown whether these two proteins alone form a complex or require additional components for interaction. Here, we demonstrate that ALKBH2 can directly interact with PCNA independent from other cellular factors, and we identify the hydrophobic pocket of PCNA as the key domain mediating this interaction. Moreover, we find that PCNA association with ALKBH2 increases significantly during DNA replication, suggesting that ALKBH2 forms a cell-cycle dependent complex with PCNA. Intriguingly, we show that an ALKBH2 germline variant, as well as a variant found in cancer, display altered interaction with PCNA. Our studies reveal the ALKBH2 binding interface of PCNA and indicate that both germline and somatic ALKBH2 variants could have cellular effects on ALKBH2 function in DNA repair.