Biomarkers to assess the targeting of DNA repair pathways to augment tumor response to therapy

Hyper-activation of DNA repair pathways can enable tumor cells to survive DNA damage. Therefore, targeting specific DNA repair pathways might prove efficacious for cancer therapy. The advent of personalized therapy necessitates novel biomarkers to assess tumor response to therapy. Biological indicators are vital in the field of cancer research and treatment. The focus of this review is on the DNA repair machinery as an emerging target for enhancement of therapy. Additionally, DNA damage and repair biomarkers for prognosis in different types of cancer will be discussed. The application of biomarkers to assess tumor response to therapy based on targeting DNA repair pathways can potentially improve patient quality of life and aid in treatment design.     

Many Ways to Loop DNA

In the 1960s, I developed methods for directly visualizing DNA and DNA-protein complexes using an electron microscope. This made it possible to examine the shape of DNA and to visualize proteins as they fold and loop DNA. Early applications included the first visualization of true nucleosomes and linkers and the demonstration that repeating tracts of adenines can cause a curvature in DNA. The binding of DNA repair proteins, including p53 and BRCA2, has been visualized at three- and four-way junctions in DNA. The trombone model of DNA replication was directly verified, and the looping of DNA at telomeres was discovered.     

p53 binds to cisplatin-damaged DNA

We have previously shown that bacterially expressed p53 protein or p53 protein isolated from cis-diamminedichloroplatinum II (cisplatin)-damaged cells is capable of binding to double-stranded platinated DNA molecules lacking any p53 DNA binding sites. Here we report using various p53 mutants that two separate domains of p53 protein affect p53 binding to platinated DNA. Mutations within the central core of p53, the domain responsible for sequence-specific DNA binding activity, completely eliminated p53 binding to platinated DNA. Based on competition experiments p53 preferred binding to sequence-specific DNA molecules over platinated DNA molecules. However, p53 binding to platinated DNA molecules was significantly stronger than p53 interactions with DNA molecules lacking damage and a p53 consensus site. Finally, an antibody specific to the C-terminal domain of p53 (pAb421) which activates sequence-specific DNA binding activity inhibited p53 binding to platinated DNA. Taken together, these results suggest that in addition to binding to p53 DNA binding sites, p53 also interacts with cisplatin-damaged DNA molecules.     

Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) ‘clamps’ for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by ‘hybrid’ phosphorothioate–LNA ODNs induces tumour necrosis factor-α production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.     

Antibodies to DNA

Antibodies that recognize specific conformational variations of DNA structure provide sensitive reagents for testing the extent to which such conformational heterogeneity occurs in nature. A most dramatic recent example has been the development and application of antibodies to left-handed Z-DNA. They provided the first identification of Z-DNA in fixed nuclei and chromosomes, and of DNA sequences that form Z-DNA under the influence of supercoiling. Antibodies have also been induced by chemically modified DNA and by synthetic polydeoxyribonucleotides that differ from the average B-DNA structure. These antibodies recognize only the features that differ from native DNA. In most experiments, native DNA itself is not immunogenic. Antibodies that do react with native DNA occur in sera of patients with autoimmune disease, but even monoclonal anti-DNA autoantibodies usually react with other polynucleotides as well. Anti-DNA antibodies, especially those of monoclonal origin, provide a model for the study of protein-nucleic acid recognition.     

Novel Strategies to Construct Complex Synthetic Vectors to Produce DNA Molecular Weight Standards

DNA molecular weight standards (DNA markers, nucleic acid ladders) are commonly used in molecular biology laboratories as references to estimate the size of various DNA samples in electrophoresis process. One method of DNA marker production is digestion of synthetic vectors harboring multiple DNA fragments of known sizes by restriction enzymes. In this article, we described three novel strategies—sequential DNA fragment ligation, screening of ligation products by polymerase chain reaction (PCR) with end primers, and “small fragment accumulation”—for constructing complex synthetic vectors and minimizing the mass differences between DNA fragments produced from restrictive digestion of synthetic vectors. The strategy could be applied to construct various complex synthetic vectors to produce any type of low-range DNA markers, usually available commercially. In addition, the strategy is useful for single-step ligation of multiple DNA fragments for construction of complex synthetic vectors and other applications in molecular biology field. Zhe Chen and Jianbing Wu contributed to this work equally.     

Histones bind more tightly to bromodeoxyuridine-substituted DNA than to normal DNA.

Using a membrane filter assay, we have obtained results from both kinetic and competition experiments indicating that histones bind more strongly to bromodeoxyuridine-substituted DNA than to normal DNA. At 37 degrees C in our standard buffer of 0.2 M ionic strength, the rate of dissociation of histones H1, H2, and h4 from BrdU-substituted DNA is respectively 7, 4, and 2 times slower than it is from normal DNA. Competition experiments show an even greater difference between BrdU-substituted and normal DNA with respect to histone binding. The tighter binding of histones to BrdU-substituted DNA is of interest because of the known effects of BrdU on eukaryotic chromosome condensation and staining, virus induction, and the inhibition of differentiation.     

Gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.     

miRNA response to DNA damage

Faithful transmission of genetic material in eukaryotic cells requires not only accurate DNA replication and chromosome distribution but also the ability to sense and repair spontaneous and induced DNA damage. To maintain genomic integrity, cells undergo a DNA damage response using a complex network of signaling pathways composed of coordinate sensors, transducers and effectors in cell cycle arrest, apoptosis and DNA repair. Emerging evidence has suggested that miRNAs play a crucial role in regulation of DNA damage response. In this review, we discuss the recent findings on how miRNAs interact with the canonical DNA damage response and how miRNA expression is regulated after DNA damage.     

Oxidative damage to plant DNA in relation to growth conditions.

In this study we investigated the level of 8-oxo-2'-deoxyguanosine (8-oxodG) in DNA of Cardamine pratensis plants subjected to different growth conditions trying to answer the question whether factors like light and water accessibility or low temperature may have an impact on the total DNA oxidative damage. The level of this modified nucleoside was determined using HPLC coupled to UV absorbance and electrochemical detection (HPLC-UV-EC). We did not observe any statistically significant differences in 8-oxodG level between DNA of etiolated and light exposed plants as well as between DNA of regularly watered and drought-subjected plants. In contrast, we have shown that chilling (1 degree C for 28 h) brings about the increase of 8-oxodG level in DNA.     

My Journey to DNA Repair

I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have discovered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases Ⅰ, and Ⅳ. I discovered the mammalian exonucleases DNase Ⅲ (TREX1) and IV (FEN1). Interestingly, expression of TREX1 was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O6-methylguanine (O6 mG) is repaired without removing the guanine from DNA, identifying a surprising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation.     

BMI1 is recruited to DNA breaks and contributes to DNA damage-induced H2A ubiquitination and repair

DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G(2)/M. These data support a crucial role for BMI1 in the cellular response to DNA damage.     

Abrogation of the CLK-2 checkpoint leads to tolerance to base-excision repair intermediates

Incorporation of uracil during DNA synthesis is among the most common types of endogenously generated DNA damage. Depletion of Caenorhabditis elegans dUTPase by RNA interference allowed us to study the role of DNA damage response (DDR) pathways when responding to high levels of uracil in DNA. dUTPase depletion compromised development, caused embryonic lethality and led to activation of cell-cycle arrest and apoptosis. These phenotypes manifested as a result of processing misincorporated uracil by the uracil-DNA glycosylase UNG-1. Strikingly, abrogation of the clk-2 checkpoint gene rescued lethality and developmental defects, and eliminated cell-cycle arrest and apoptosis after dUTPase depletion. These data show a genetic interaction between UNG-1 and activation of the CLK-2 DDR pathway after uracil incorporation into DNA. Our results indicate that persistent repair intermediates and/or single-stranded DNA formed during repair of misincorporated uracil are tolerated in the absence of the CLK-2 checkpoint in C. elegans.     

Damage to DNA and activity of nuclear DNA repair and replicative enzymes following N-nitrosodiethylamine treatment to rats

Continuous administration in the drinking water of hepatocarcinogen N-nitrosodiethylamine (NDEA) to male rats (200 mg/L) for 60 days resulted in DNA damage in the form of single strand breaks. The damage, which is measured as a shift in the sedimentation of DNA in alkaline sucrose density gradients, was found to be maximum at the fourth week of treatment, and the sedimentation pattern of DNA was found to return to near normal size by the seventh week of NDEA treatment. Simultaneously, there were perturbations in the nuclear enzymes involved in DNA replication and repair. Activities of DNA polymerase beta, DNA ligase, and topoisomerase were found to increase in as early as the first week of NDEA treatment and reached the maximum at the fourth week, and thereafter declined to normal level by the eighth week of treatment. Concomitantly, the activities of DNA polymerase alpha, DNA primase, and RNA polymerase which were unaltered in the initial period of carcinogen treatment recorded a marked increase after sixth week of NDEA treatment. Results suggest that administration of NDEA inflicts DNA damage, which is manifested as increase in DNA repair enzymes in the initial period and activated DNA replicative enzymes at a later period, indicating the active proliferation of transformed cells.     

Photoaddition of chlorpromazine to DNA

Chlorpromazine, 2-chloro-N-(3-dimethylaminopropyl)phenothiazine (CPZ), is a frequently prescribed antipsychotic drug that causes cutaneous photosensitivity in man. CPZ is also phototoxic and photomutagenic in vitro. We have investigated the photoaddition of CPZ to DNA as a possible mechanism for these photobiologic effects. Prior to irradiation, CPZ binds non-covalently to double-stranded calf thymus DNA. At high nucleotide to CPZ ratios, the CPZ absorption maximum shifts from 305 nm to 340 nm with an isosbestic point at 323 nm and 90% of the CPZ fluorescence at 455 nm is quenched. The excitation and emission spectra for the unquenchable fluorescence are the same as those for unbound CPZ. The absorption and fluorescence spectra of unbound CPZ are restored at 0.1 mM magnesium acetate or 100 mM sodium acetate. Non-covalent binding of CPZ to heat-denatured DNA does not shift the CPZ absorption spectrum but quenches 65% of the CPZ fluorescence. Photolytic decomposition of CPZ was inhibited by binding to DNA. In the presence of high concentrations of double-stranded DNA or denatured DNA the photolysis rates were reduced by greater than 98% and 65%, respectively, compared to free CPZ. Formation of covalent photoadducts between CPZ and denatured DNA was 10-fold more efficient than photoadduct formation with double-stranded DNA. Approximately 10% of the CPZ which photodecomposed upon irradiation at 323 nm photoadded to denatured DNA. These results indicate that formation of a complex between CPZ and double-stranded DNA absorbing at 340 nm protects CPZ from photodecomposition and inhibits covalent photoadduct formation.     

How to get from A to B: strategies for analysing protein motion on DNA

Essentially all genetic events require proteins to move from one location in a DNA polymer to another location in the same chain. A protein will seldom bind to a specific site in the DNA by colliding directly with that site. Instead, the protein will almost always collide first with a random site anywhere in the DNA and then migrate to the specific site by a facilitated-diffusion process that is constrained to the zone of that DNA molecule. Thereafter, many proteins bound to their target sites translocate in a specified direction along the DNA by a energy-dependent vectorial mechanism. This review will discuss some of the strategies that have been developed to analyse the motion of proteins on DNA, with respect to both the random diffusion processes involved in target-site location by DNA-binding proteins and the vectorial processes involved in unidirectional translocation along DNA.     

Chromatin plasticity in response to DNA damage: The shape of things to come

DNA damage poses a major threat to cell function and viability by compromising both genome and epigenome integrity. The DNA damage response indeed operates in the context of chromatin and relies on dynamic changes in chromatin organization. Here, we review the molecular bases of chromatin alterations in response to DNA damage, focusing on core histone mobilization in mammalian cells. Building on our current view of nucleosome dynamics in response to DNA damage, we highlight open challenges and avenues for future development. In particular, we discuss the different levels of regulation of chromatin plasticity during the DNA damage response and their potential impact on cell function and epigenome maintenance.     

Genomic approaches to DNA repair and mutagenesis

DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways.     

A real-time QCM-D approach to monitoring mammalian DNA damage using DNA adsorbed to a polyelectrolyte surface

We have successfully demonstrated that the quartz crystal microbalance with dissipation monitoring (QCM-D) can be used to monitor real-time damage to genomic mammalian DNA adsorbed to a polyelectrolyte surface. To reveal the capabilities of this technique, we exposed DNA surfaces to quercetin, an agent that has been implicated in causing DNA strand breaks in a Cu(II)-dependent fashion in vitro. We show that the QCM-D frequency and dissipation patterns that result from exposure of the DNA surfaces to quercetin-Cu(II) are consistent with the induction of DNA strand scission. We use QCM-D to furthermore demonstrate that this process is dependent on Cu(II) and that the DNA damage induced by quercetin can still be detected if Cu(II) is in situ with the DNA surface and not in solution phase.