Covalent binding between proteins and a DNA strand produces DNA-protein crosslinks (DPC). DPC are one of the most deleterious types of DNA damage, leading to the blockage of DNA replication and transcription. Both DNA lesions and endogenous products with carbonyl functional groups can produce DPC in genomic DNA under normal physiological conditions. For example, formaldehyde, the most abundant endogenous human carcinogen, and apurinic/apyrimidinic (AP) sites, the most common type of endogenous DNA lesions, has been shown to crosslink proteins and/or DNA through their carbonyl functional groups. Unfortunately, compared to other types of DNA damage, DPC have been less studied and understood. However, a recent advancement has allowed researchers to determine accurate yields of various DNA lesions including formaldehyde-derived DPC with high sensitivity and specificity, paving the way for new developments in this field of research. Here, we review the current literature and remaining unanswered questions on DPC formation by endogenous formaldehyde and various aldehydic 2-deoxyribose lesions.

Doxorubicin has been in use as a key anticancer drug for forty years, either as a single agent or in combination chemotherapy. It functions primarily by interfering with topoisomerase II activity but in the presence of formaldehyde, it forms adducts with DNA, mainly with the exocyclic amine of guanine at GpC sites and these adducts are more cytotoxic than topoisomerase II induced damage. High levels of adducts form spontaneously from the endogenous level of formaldehyde in tumour cells (1,300 adducts per cell after a 4 hr treatment with doxorubicin), but substantially higher levels form with the addition of exogenous sources of formaldehyde, such as formaldehyde releasing prodrugs. The enhanced cytotoxicity of adducts has been confirmed in mouse models, with adduct-forming conditions resulting in much improved inhibition of tumour growth, as well as cardioprotection. Doxorubicin cardiotoxicity has been attributed to topoisomerase II poisoning, and the cardioprotection is consistent with a mechanism switch from topoisomerase II poisoning to covalent adduct formation. Although the adducts have a half-life of less than one day, a population remains as essentially permanent lesions. The capacity of doxorubicin to form adducts offers a range of potential advantages over the conventional use of doxorubicin (as a topoisomerase II poison), including: enhanced cell kill; tumour-selective activation, hence tumour-selective cell kill; decreased cardiotoxicity; decreased resistance to prolonged doxorubicin treatment. There is therefore enormous potential to improve clinical responses to doxorubicin by using conditions which favour the formation of doxorubicin-DNA adducts.

Interactions of DNA with structural proteins such as histones, regulatory proteins and enzymes play a crucial role in major cellular processes such as transcription, replication and repair. The in vivo mapping and characterization of the binding sites of the involved biomolecules are of primary importance for a better understanding of genomic deployment that is implicated in tissue and developmental stage‐specific gene expression regulation. The most powerful and commonly used approach to date is immunoprecipitation of chemically cross‐linked chromatin (XChIP) coupled with sequencing analysis (ChIP‐seq). While the resolution and the sensitivity of the high‐throughput sequencing techniques have been constantly improved, little progress has been achieved in the cross‐linking step. Because of its low efficiency, the use of the conventional UVC lamps remains very limited while the formaldehyde method was established as the “gold standard” cross‐linking agent. Efficient biphotonic cross‐linking of directly interacting nucleic acid–protein complexes by a single short UV laser pulse has been introduced as an innovative technique for overcoming limitations of conventionally used chemical and photochemical approaches. In this survey, the main available methods including the laser approach are critically reviewed for their ability to generate DNA–protein cross‐links in vitro model systems and cells.

Doxorubicin (DOX) is a widely used classical broad-spectrum anti-cancer drug. The major mechanism of DOX-mediated anticancer activity at clinically relevant concentrations is believed to be via DNA double-strand breaks due to topoisomerase IIα. However, other mechanisms by which DOX causes cytotoxicity have been proposed, including formaldehyde-dependent virtual interstrand cross-linking (ICL) formation. In this study, a method was established whereby cytotoxicity caused by virtual ICL derived from DOX is turned on and off using a cell culture system. Using this strategy, DOX-mediated cytotoxicity in Fanconi anemia group gene (FANC)/breast cancer susceptibility gene (BRCA)-deficient cells increased up to 70-fold compared to that in cells proficient in DNA repair pathways by increasing intracellular formaldehyde (FA) concentration. This approach also demonstrated that cytotoxicity introduced by DOX-mediated FA-dependent virtual ICL is completely independent of the toxicity induced by topoisomerase II inhibition at the cellular level. The potential of dual-targeting by DOX treatment was verified using an acid-specific FA donor. Overall, anticancer therapy targeting tumors deficient in the FANC/BRCA pathway may be possible by minimizing DOX-induced toxicity in normal cells.

Plants are frequently exposed to adverse conditions. Priming, also known as acclimation or hardening, induces stress memory and prepares plants for future challenges by activating defense and protective mechanisms. For this reason, priming is an effective means to maintain plant yield in the face of climate change. Memory behind the priming is mainly based on epigenetic modifications, for example, histone posttranslational modifications (PTMs) on the priming-related genes. While histone PTMs are the most diverse group of epigenetic modifications and regulate gene expression via addition of chemical groups to histone amino acids, their characterization is challenging. Chromatin immunoprecipitation (ChIP) is an essential method for the characterization of histone PTMs; however, subject to many challenges, especially in plant samples. This review discusses the current understanding of histone modifications in plant stress and priming and the ChIP methodology and troubleshooting. Yield losses resulting from climate change necessitate the use of priming as an agricultural practice. In order to apply priming, an in-depth analysis of stress- or priming-induced histone PTMs is essential. ChIP has been extensively used in plant stress studies and has undergone numerous improvements. Although there are more sophisticated methods, ChIP is still regarded as a standard method for the characterization of chromatin profiles. This review aims to support researchers in the utilization of ChIP, particularly, for plant stress and/or priming studies.

Formaldehyde (FA) is a mutagenic chemical - a property mitigated in vivo by rapid detoxification and limited tissue distribution following inhalation of the free agent. Endogenously produced FA is necessary for life and required for one-carbon transfer reactions; however, FA derived from external sources (exogenous FA), which may be in the form of methanol, may increase in vivo concentrations above naturally occurring physiological levels. Both endogenous and exogenous FA produce DNA monoadducts, DNA-DNA and DNA-protein cross-links (DDX and DPX) but, when exposed to exogenously-derived free FA, DNA monoadducts, DDX, and DPX are only produced at initial sites of contact. In contrast, methanol may systemically induce DNA adducts distally. FA also induces oxidative stress/lipid peroxidation with some individuals suggesting the resulting reactive aldehydes may have the potential to induce distal site DNA damage with the resulting reactive aldehydes having the potential to induce distal site DNA damage. Chromosome changes in the form of aberrations or micronuclei in blood cells have been studied in FA-exposed animals and humans, with most of the former being negative. Human occupational studies have given mixed results for such changes in peripheral blood lymphocytes (PBLs) which circulate widely but do not reflect recent bone marrow (BM) events. Recent studies reporting changes in human BM or hematopoietic precursor cells (HPCs) either have had confounding exposures or could not distinguish in vivo from in vitro occurrences. The reported genetic changes in circulating blood cells do not provide convincing support for FA's classification as a human leukemogen.

Chromatin-immunoprecipitation (ChIP) employs generally a mild formaldehyde cross-linking step, which is followed by isolation of specific protein-DNA complexes and subsequent PCR testing, to analyze DNA-protein interactions. Poly(ADP-ribosyl)ation, a posttranslational modification involved in diverse cellular functions like repair, replication, transcription, and cell death regulation, is most prominent after DNA damage. Poly(ADP-ribose)polymerase-1 is activated upon binding to DNA strand-breaks and coordinates repair by recruitment or displacement of proteins. Several proteins involved in different nuclear pathways are directly modified or contain poly(ADP-ribose)-interaction motifs. Thus, poly(ADP-ribose) regulates chromatin composition. In immunofluorescence experiments, we noticed artificial polymer-formation after formaldehyde-fixation of undamaged cells. Therefore, we analyzed if the formaldehyde applied during ChIP also induces poly(ADP-ribosyl)ation and its impact on chromatin composition. We observed massive polymer-formation in three different ChIP-protocols tested independent on the cell line. This was due to induction of DNA damage signaling as monitored by γH2AX formation. To abrogate poly(ADP-ribose) synthesis, we inhibited this enzymatic reaction either pharmacologically or by increased formaldehyde concentration. Both approaches changed ChIP-efficiency. Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well

Transcription of a gene can be regulated at many different levels. One such fundamental level is interaction between protein and DNA. Protein(s) binds to distinct sites on the DNA, which activate, enhance or repress transcription. Despite being such an important process, very few tools exist to identify the proteins that interact with chromosome, most of which are in vitro in nature. Here, we propose an in vivo based method for identification of DNA binding protein(s) in bacteria where the DNA-protein complex formed in vivo is crosslinked by formaldehyde. This complex is further isolated and t

highly permeable to formaldehyde. Formaldehyde cross-linking is frequently employed for the detection and quantification of DNA-protein and protein-protein Hi-C is a high-throughput genomic and epigenomic technique to capture chromatin conformation (3C). In general, Hi-C is considered as a derivative of a series of chromosome conformation capture technologies, including but not limited to 3C (chromosome conformation capture), 4C (chromosome conformation capture-on-chip/circular chromosome conformation capture), and 5C (chromosome conformation capture Cell and nuclear membranes are highly permeable to formaldehyde. Formaldehyde cross-linking is frequently employed for the detection and quantification of DNA-protein and protein-protein interactions. Of interest in the context of Hi-C, and all 3C-based methods, is the ability of formaldehyde to capture cis chromosomal interactions between distal segments of chromatin. It does so by forming covalent links between spatially adjacent chromatin segments. Formaldehyde can react with macromolecules in two steps: first it reacts with a nucleophilic group on a DNA base for example, and forms a methylol adduct, which is then converted to a Schiff base. In the second step, the Schiff base, which can decompose rapidly, forms a methylene bridge with another functional group on another molecule. It can also make this methylene bridge with a small molecule in solution such as glycine, which is used in excess to quench formaldehyde in Hi-C. Quenchers can typically exert an effect on formaldehyde from outside the cell. A key feature of this two-step formaldehyde crosslinking reaction is that all the reactions are reversible, which is vital for chromatin capture. Crosslinking is a pivotal step of the chromatin capture workflow as the functional readout of the technique is the frequency at which two genomic regions are crosslinked to each other. Thus, the standardization of this step is important and for that, one must consider potential sources of variation. Presence of serum, which contains a high concentration of protein, in culture media can decrease the effective concentration of formaldehyde available for chromatin crosslinking, by sequestering it in the culture media. Therefore, in cases where serum is used in culture, it should be removed for the crosslinking step. The nature of cells, i.e., whether they are suspension or adherent, is also a pertinent consideration for the crosslinking step. Adherent cells bind to surfaces with the help of molecular mechanisms of cytoskeletons. It has been shown that there is a link between cytoskeleton-maintained nuclear and cellular morphology which, if altered, may negatively impact global nuclear organization. Adherent cells…

Ulm, Universität Ulm, Diss., 2000