Ingrid Tessmer

Modulating ferroptosis, a form of cell death driven by uncontrolled lipid peroxidation, is of interest in numerous diseases. Here, we found that the deletion of phosphoglycolate phosphatase (PGP), an essential enzyme that safeguards high glycolytic flux, suppresses ferroptosis. Using metabolomic and isotopic labeling experiments together with lipid and proteomic profiling, we find that PGP loss drives a rewiring of the pentose phosphate pathway and of cellular energy and lipid metabolism that triggers a multifactorial antioxidant response. Paradoxically, our attempts to block PGP pharmacologically led to the realization that the recently described PGP inhibitor compound 1 (CP1) exerts a strong ferroptosis-sensitizing effect. Using genetic, biochemical, and biophysical approaches, we characterize CP1 as a direct, species-independent, dual inhibitor of PGP and ferroptosis suppressor protein 1 (FSP1), and further find that CP1 triggers FSP1 self-assembly. In sum, we identify PGP as a target protein for ferroptosis control and introduce a small-molecule FSP1 inhibitor with unique features to the armamentarium of pharmacological ferroptosis modulators.

The intricate interplay between DNA and proteins is key for biological functions such as DNA replication, transcription and repair. Dynamic nanoscale observations of DNA structural features are necessary for understanding these interactions. Here we introduce graphene energy transfer with vertical nucleic acids (GETvNA), a method to investigate DNA–protein interactions that exploits the vertical orientation adopted by double-stranded DNA on graphene. This approach enables the dynamic study of DNA conformational changes via energy transfer from a probe dye to graphene, achieving spatial resolution down to the Ångström scale at subsecond temporal resolution. We measured DNA bending induced by adenine tracts, bulges, abasic sites and the binding of endonuclease IV. In addition, we observed the translocation of the O⁶-alkylguanine DNA alkyltransferase on DNA, reaching single base-pair resolution and detecting preferential binding to adenine tracts. This method promises widespread use for dynamical studies of nucleic acids and nucleic acid–protein interactions with resolution so far reserved for traditional structural biology techniques.

Given life’s dependence on genome maintenance, unsurprisingly, investigations of the molecular processes involved in protecting the genome or, failing this, repairing damages to and alterations introduced into genetic material are at the forefront of current research

DNA alkyltransferase and alkyltransferase-like family proteins are responsible for the repair of highly mutagenic and cytotoxic O⁶-alkylguanine and O⁴-alkylthymine bases in DNA. Their mechanism involves binding to the damaged DNA and flipping the base out of the DNA helix into the active site pocket in the protein. Alkyltransferases then directly and irreversibly transfer the alkyl group from the base to the active site cysteine residue. In contrast, alkyltransferase-like proteins recruit nucleotide excision repair components for O⁶-alkylguanine elimination. One or more of these proteins are found in all kingdoms of life, and where this has been determined, their overall DNA repair mechanism is strictly conserved between organisms. Nevertheless, between species, subtle as well as more extensive differences that affect target lesion preferences and/or introduce additional protein functions have evolved. Examining these differences and their functional consequences is intricately entwined with understanding the details of their DNA repair mechanism(s) and their biological roles. In this review, we will present and discuss various aspects of the current status of knowledge on this intriguing protein family.

The intricate interplay between DNA and proteins is key for biological functions such as DNA replication, transcription, and repair. To better understand these interactions, it is crucial to develop tools to study DNA-protein complexes with high spatial and temporal resolution. Here, we use the vertical orientation that DNA adopts on graphene and investigate its interactions with proteins via energy transfer from a probe dye to graphene, achieving spatial resolution down to the Ångström scale. We measured the bending angle of DNA induced by adenine tracts, bulges, abasic sites and the binding of Escherichia coli endonuclease IV with unprecedented precision and millisecond time resolution. Additionally, we observed the translocation of the O ⁶ -alkylguanine DNA alkyltransferase along double-stranded DNA, reaching single-base pair resolution and detecting an affinity for adenine tracts. Overall, we foresee that this method will become a widespread tool for the dynamical study of nucleic acid and nucleic acid-protein interactions.

The base excision repair (BER) glycosylase hOGG1 (human oxoguanine glycosylase 1) is responsible for repairing oxidative lesions in the genome, in particular oxidised guanine bases (oxoG). In addition, a role of hOGG1 in transcription regulation by recruitment of various transcription factors has been reported. Here, we demonstrate direct interactions between hOGG1 and the medically important oncogene transcription factor Myc that is involved in transcription initiation of a large number of genes including inflammatory genes. Using single molecule atomic force microscopy (AFM), we reveal recruitment of Myc to its E-box promoter recognition sequence by hOGG1 specifically under oxidative stress conditions, and conformational changes in hOGG1-Myc complexes at oxoG lesions that suggest loading of Myc at oxoG lesions by hOGG1. Importantly, our data show suppression of hOGG1 catalytic activity in oxoG repair by Myc. Furthermore, mutational analyses implicate the C28 residue in hOGG1 in oxidation induced protein dimerisation and suggest a role of hOGG1 dimerisation under oxidising conditions in hOGG1-Myc interactions. From our data we develop a mechanistic model for Myc recruitment by hOGG1 under oxidising, inflammatory conditions, which may be responsible for the observed enhanced gene expression of Myc target genes.

Significance: We directly visualize DNA translocation and lesion recognition by the O ⁶ -alkylguanine DNA alkyltransferase (AGT). Our data show bidirectional movement of AGT monomers and clusters on undamaged DNA that depended on Zn ²⁺ occupancy of AGT. A role of cooperative AGT clusters in enhancing lesion search efficiencies by AGT has previously been proposed. Surprisingly, our data show no enhancement of DNA translocation speed by AGT cluster formation, suggesting that AGT clusters may serve a different role in AGT function. Our data support preferential cluster formation by AGT at alkyl lesions, suggesting a role of these clusters in stabilizing lesion-bound complexes. From our data, we derive a new model for the lesion search and repair mechanism of AGT.

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α‐primase (Pol‐prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single‐stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA‐bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol‐prim on free and RPA‐bound ssDNA. Atomic force microscopy imaging showed that while Tag_131‐627(V350E/P417D) and Tag_131‐627(L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full‐length Tag SV40 Tag_1‐708 and monomeric M2 stimulated DNA synthesis of Pol‐prim on ssDNA and on RPA‐bound ssDNA. In contrast, neither monomeric M1 nor M1‐M2 dimers could stimulate Pol‐prim, on ssDNA or on RPA‐bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1‐M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein–protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation.

DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA.