The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.

DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification.

The Mre11 nuclease is involved in early responses to DNA damage, often mediated by its role in DNA end processing. MRE11 mutations and aberrant expression are associated with carcinogenesis and cancer treatment outcomes. While, in recent years, progress has been made in understanding the role of Mre11 nuclease activities in DNA double-strand break repair, their role during replication has remained elusive. The nucleoside analog gemcitabine, widely used in cancer therapy, acts as a replication chain terminator; for a cell to survive treatment, gemcitabine needs to be removed from replicating DNA. Activities responsible for this removal have, so far, not been identified. We show that Mre11 3' to 5' exonuclease activity removes gemcitabine from nascent DNA during replication. This contributes to replication progression and gemcitabine resistance. We thus uncovered a replication-supporting role for Mre11 exonuclease activity, which is distinct from its previously reported detrimental role in uncontrolled resection in recombination-deficient cells.

U–G mismatches in DNA generally result from spontaneous cytosine deamination and are repaired by the base excision repair pathway to avoid mutations. However, as part of the adaptive immune response in B cells U–G mismatches are enzymatically generated in the Ig genes by activation-induced cytidine deaminase. These uracils are further processed by the uracil–DNA glycosylase UNG and funneled into mutagenic pathways to generate point mutations and strand breaks resulting in somatic hypermutation and class shift recombination, respectively. Cytosine deamination by activation-induced cytidine deaminase and other DNA deaminases is also a significant source of mutations in cancer. Human DNA polymerases do not discriminate between dTTP and dUTP. Misincorporation of dUMP generates U–A pairs that are indirectly mutagenic through erroneous processing of abasic sites. Some cells have surprisingly high dUTP/dTTP ratios, suggesting that the incorporation of dUMP during DNA replication or repair may be considerable. Mammalian cells have four different uracil–DNA glycosylases; UNG, SMUG1, TDG and MBD4. These have in part complementary, but also distinct functions in base excision repair, immunity and epigenetic regulation. In addition, they may modify responses to fluoropyrimidines in cancer treatment.

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

UNG is the major uracil-DNA glycosylase in mammalian cells and is involved in both error-free base excision repair of genomic uracil and mutagenic uracil-processing at the antibody genes. However, the regulation of UNG in these different processes is currently not well understood. The UNG gene encodes two isoforms, UNG1 and UNG2, each possessing unique N-termini that mediate translocation to the mitochondria and the nucleus, respectively. A strict subcellular localization of each isoform has been widely accepted despite a lack of models to study them individually. To determine the roles of each isoform, we generated and characterized several UNG isoform-specific mouse and human cell lines. We identified a distinct UNG1 isoform variant that is targeted to the cell nucleus where it supports antibody class switching and repairs genomic uracil. We propose that the nuclear UNG1 variant, which in contrast to UNG2 lacks a PCNA-binding motif, may be specialized to act on ssDNA through its ability to bind RPA. RPA-coated ssDNA regions include both transcribed antibody genes that are targets for deamination by AID and regions in front of the moving replication forks. Our findings provide new insights into the function of UNG isoforms in adaptive immunity and DNA repair.

DNA binding as an anti-inflammatory Mice that lack the gene encoding 8-oxoguanine DNA glycosylase 1 (OGG1) show resistance to inflammation. This enzyme binds to sites of oxidative DNA damage and initiates DNA base excision repair. Visnes et al. developed a small-molecule drug that acts as a potent and selective active-site inhibitor that stops OGG1 from recognizing its DNA substrate (see the Perspective by Samson). The drug inhibited DNA repair and modified OGG1 chromatin dynamics, which resulted in the inhibition of proinflammatory pathway genes. The drug was well tolerated by mice and suppressed lipopolysaccharide- and tumor necrosis factor–α–mediated neutrophilic inflammation in the lungs. Science , this issue p. 834 ; see also p. 748

Genomic Uracil, pp. 1-13 (2018) Free AccessChapter 1: Introduction — DNA Repair is Integrated with Many Cellular ProcessesHans E. Krokan, Bodil Kavli and Geir SlupphaugHans E. Krokan, Bodil Kavli and Geir Slupphaughttps://doi.org/10.1142/9789813233508_0001Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Long-term survival of a species requires cellular mechanisms that efficiently safeguard DNA, the chemical material of the genome. All known living organisms have several mechanisms that repair different types of DNA damage. This is essential to prevent cytotoxic and mutagenic effects of damage to DNA. In fact, each cell in the body is inflicted by tens of thousands of DNA lesions per day from spontaneous chemical decay and normal cellular metabolites alone. Environmental chemicals, ultraviolet and ionizing radiation add to the burden of genome damage. Without cellular mechanisms that maintain DNA, we would probably die within a few days from multiple organ failure due to inactivation of numerous genes in each cell. Fortunately, a number of mechanisms contribute to maintaining DNA, including high fidelity DNA replication, DNA repair, cell cycle regulation, as well as removal of cells with serious genetic defects by apoptosis (programmed cell death) (Fig. 1.1). However, this is just the tip of the iceberg, as outlined below. FiguresReferencesRelatedDetails Genomic UracilMetrics Downloaded 200 times History PDF download

Genomic Uracil, pp. 127-152 (2018) No AccessChapter 5: Viral Uracil — Uracil DNA Glycosylases and dUTPasesHans E. Krokan, Bodil Kavli, and Geir SlupphaugHans E. Krokan, Bodil Kavli, and Geir Slupphaughttps://doi.org/10.1142/9789813233508_0005Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: DNA viruses and retroviruses use various strategies to avoid and remove genomic uracil. Similar to the case for cells, dUTPase and UNG-type uracil-DNA glycosylase are central in these processes. Large DNA viruses encode viral variants of Family 1 (UNG) enzymes, while smaller viruses depend solely on host cell enzymes. Genes for viral dUTPase and UNG are among those that are expressed early after infection. Herpesviridae and poxviridae encode their own uracil-DNA glycosylase, dUTPase and DNA polymerase. Midsize DNA viruses in the adenoviridae family encode their own DNA polymerase, but not uracil-DNA glycosylase. Fowl adenovirus 9 also encodes a dUTPase, but in human adenoviruses a dUTPase-like gene has diverged to become a transforming factor named E4-ORF1. Retroviridae do not encode uracil-DNA glycosylase, but some of them capture host uracil-DNA glycosylase, which is encapsulated in the virus particle and released after infection. Furthermore, several major retroviruses encode dUTPase, although this is not the case for primate lentiviruses that instead use captured UNG to minimize genomic uracil in replicative DNA intermediates. Herpesvirus uracil-DNA glycosylase has strong homology to E. coli, yeast and mammalian UNG proteins, while Poxvirus uracil-DNA glycosylase is a more distant member of the same family. Herpesviruses and adenoviruses replicate in the host nucleus, where they in part can rely on host factors. In contrast, poxviruses replicate in replication factories in the cytosol and encode several DNA replication and repair proteins as well as nucleotide metabolizing enzymes. Interestingly, while herpesvirus and poxvirus UNG proteins are genuine uracil-DNA glycosylases, they also have important functions in viral replication. This has been extensively studied for poxvirus UNG, which is essential for virus replication. Poxvirus UNG (D4) forms a heterotrimeric complex with an adapter protein (A20) and the pox DNA polymerase (E9) and acts as a polymerase processivity factor. Herpes UNG is particularly important for virulence in nerve cells, which lack or have very low levels of cellular UNG. The function of host UNG in retroviridae is complex, as outlined below. The special functions of viral UNG and dUTPase make these proteins potential drug targets. The giant mimivirus Acanthamoeba polyphaga (see Chapter 2) encodes numerous DNA repair proteins, including an UNG-type uracil-DNA glycosylase. Its sequence and crystal structure demonstrate the presence of a catalytic domain containing structural and functional motifs typically present in other UNG proteins and an unstructured N-terminal extension typical of mammalian UNG proteins. FiguresReferencesRelatedDetails Genomic UracilMetrics History PDF download

Genomic Uracil, pp. 275-286 (2018) No AccessChapter 9: Genomic Uracil — Valuable Tool in Molecular Biology but Inherent Problem in Sequencing of Ancient DNAGeir Slupphaug and Hans E. KrokanGeir Slupphaug and Hans E. Krokanhttps://doi.org/10.1142/9789813233508_0009Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Uracil holds a rather unique position among the non-canonical DNA bases in that it can be formed both enzymatically and spontaneously in DNA from cytosine. It can also be introduced via dUTP by DNA polymerases. Furthermore, it can be excised by DNA glycosylases to initiate DNA strand cleavage and nucleotide replacement. These characteristics provide opportunities to exploit uracil as a tool within molecular biology and medicine, but also represent inherent problems regarding DNA analysis, especially when sequencing ancient DNA. The following chapter will describe some analytical methods in which DNA-uracil constitutes an important intermediate. Finally, inherent problems associated with spontaneous cytosine deamination will be discussed in the context of analysis of stored archive samples and ancient DNA. FiguresReferencesRelatedDetails Genomic UracilMetrics History PDF download

Genomic Uracil, pp. 89-126 (2018) No AccessChapter 4: Enzymology of Genomic Uracil RepairHans E. Krokan, Bodil Kavli, Antonio Sarno, and Geir SlupphaugHans E. Krokan, Bodil Kavli, Antonio Sarno, and Geir Slupphaughttps://doi.org/10.1142/9789813233508_0004Cited by:2 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Whereas several laboratories contributed to the discovery of other DNA excision repair processes, the discovery of base excision repair (BER) can be uniquely ascribed to the work of Tomas Lindahl (Fig. 4.1). He quantified spontaneous deamination of cytosine to uracil in genomic DNA and reasoned that there had to be a mecshanism correcting the highly mutagenic U:G mismatches resulting from the deamination. The search for such a mechanism resulted in the surprising discovery of uracil-DNA glycosylase in 1974, the first enzyme in a new family of DNA repair proteins. The new enzyme was initially named 'uracil N-glycosidase,' but the name was later changed to uracil-DNA glycosylase to comply with current nomenclature. In addition to his work on DNA-cytosine deamination, Lindahl made important discoveries on other aspects of DNA instability, including rates of depurination, depyrimidination and chain breaks at abasic sites… FiguresReferencesRelatedDetailsCited By 2Base Excision Repair in Sugarcane – A New OutlookNathalia Maíra Cabral de Medeiros and Katia Castanho Scortecci23 June 2021HDACi mediate UNG2 depletion, dysregulated genomic uracil and altered expression of oncoproteins and tumor suppressors in B- and T-cell linesTobias S. Iveland, Lars Hagen, Animesh Sharma, Mirta M. L. Sousa and Antonio Sarno et al.7 April 2020 | Journal of Translational Medicine, Vol. 18, No. 1 Genomic UracilMetrics History PDF download

Nat. Genet. 46, 736–741 (2014); published online 1 June 2014; corrected after print 23 January 2017 In the version of this article initially published, the name of author Florence Le Calvez-Kelm appeared incorrectly as Florence LeCalvez-Kelm. The error has been corrected in the HTML and PDF versionsof the article.

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/- mice. We found that 5-hydroxymethyluracil accumulated in Smug1 -/- tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1 -/- mice did not accumulate uracil in their genome and Ung -/- mice showed slightly elevated uracil levels. Contrastingly, Ung -/- Smug1 -/- mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.

Well-known epigenetic DNA modifications in mammals include the addition of a methyl group and a hydroxyl group to cytosine, resulting in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) respectively.In contrast, the abundance and the functional implications of these modifications in invertebrate model organisms such as the honey bee (Apis mellifera) and the fruit fly (Drosophila melanogaster) are not well understood.Here we show that both adult honey bees and fruit flies contain 5mC and also 5hmC.Using a highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) technique, we quantified 5mC and 5hmC in different tissues of adult honey bee worker castes and in adult fruit flies.A comparison of our data with reports from human and mouse shed light on notable differences in 5mC and 5hmC levels between tissues and species.

Photodynamic therapy (PDT) is a highly selective two-step cancer treatment involving a photosensitizer and illumination with visible light in the presence of molecular oxygen. PDT is clinically approved worldwide for treating several premalignant conditions and cancer forms, especially endoscopically accessible tumors and dermatological malignancies. PDT-mediated cytotoxicity takes place via autophagy, apoptosis and necrosis, but the exact trigger mechanisms for various death-pathways are still unknown. PDT induces reactive oxygen species (ROS) through photochemical reactions. ROS can react with different macromolecules resulting in cellular damage, including oxidation of proteins. One of the known protein modifications is reversible oxidation of cysteine thiols (-SH), which in many cases constitute a redox switch to modulate protein activity and cellular signaling. Here we have examined the role of reversible oxidation of protein thiols as a potential mediator of cytotoxicity after hexylaminolevulinate-mediated photodynamic treatment (HAL-PDT) in the human epidermoid carcinoma cell line A431. Nearly 2300 proteins were found to be reversibly oxidized after HAL-PDT, of which 374 high-confidence proteins were further allocated to cellular compartments and functional networks. 115 of the high confidence proteins were associated with apoptosis and 257 have previously not been reported to be reversibly oxidized on cysteines. We find an enrichment of DNA damage checkpoint and oxidative stress response proteins. Many of these constitute potential signaling hubs in apoptosis, including ATM, p63, RSK1 p38, APE1/Ref-1 and three 14-3-3 family members. Our study represents the first comprehensive mapping of reversibly oxidized proteins subsequent to HAL-PDT. Several of the proteins constitute potentially novel redox-regulated apoptotic triggers as well as potential targets for adjuvants that may improve the efficacy of HAL-PDT and PDT using other photosensitizers.

Genes encoding pectic enzymes were introduced to wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing β-galactosidase (β-Gal-14 mutant) or rhamnogalacturonan lyase (RGL-18 mutant). After sequential extraction, β-Gal-14 hot buffer-soluble solids (HBSS) of pectin contained 54% less galactose than Karnico HBSS, representing shorter galactan side chains. The individual pectin populations of β-Gal-14 HBSS showed different modifications extended to the two sub-populations as obtained by ion-exchange chromatography. Compared to wild-type, RGL-18 HBSS contained 27% more galacturonic acid and 55% less Gal on fresh potato weight basis, which was due to the removal of galactan-rich rhamnogalacturonan I (RG-I) segments. All pectin populations of RGL-18 showed consistently low levels of RG-I segments. Transgenic modification showed side effects on the methyl-esterification and acetyl substitution of RGL-18 HBSS (DM = 53, DA = 21), but not of the β-Gal-14 HBSS in comparison to wild-type (DM = 29, DA = 54).