Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.

Chemotherapeutic compounds targeting the DNA double-strand break repair pathways : the good, the bad, and the promising

The repair of DNA double-strand breaks (DSBs) is a critical cellular mechanism that exists to ensure genomic stability. DNA DSBs are the most deleterious type of insult to a cell’s genetic material and can lead to genomic instability, apoptosis, or senescence. Incorrectly repaired DNA DSBs have the potential to produce chromosomal translocations and genomic instability, potentially leading to cancer. The prevalence of DNA DSBs in cancer due to unregulated growth and errors in repair opens up a potential therapeutic window in the treatment of cancers. The cellular response to DNA DSBs is comprised of two pathways to ensure DNA breaks are repaired: homologous recombination and non-homologous end joining. Identifying chemotherapeutic compounds targeting proteins involved in these DNA repair pathways has shown promise as a cancer therapy for patients, either as a monotherapy or in combination with genotoxic drugs. From the beginning, there have been a number of chemotherapeutic compounds that have yielded successful responses in the clinic, a number that have failed (CGK-733 and iniparib), and a number of promising targets for future studies identified. This review looks in detail at how the cell responds to these DNA DSBs and investigates the chemotherapeutic avenues that have been and are currently being explored to target this repair process.

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

KRAB zinc-finger proteins localise to novel KAP1-containing foci that are adjacent to PML nuclear bodies

The KRAB-zinc finger proteins (KRAB-ZFPs) represent a very large, but poorly understood, family of transcriptional regulators in mammals. They are thought to repress transcription via their interaction with KRAB-associated protein 1 (KAP1), which then assembles a complex of chromatin modifiers to lay down histone marks that are associated with inactive chromatin. Studies of KRAB-ZFP/KAP1-mediated gene silencing, using reporter constructs and ectopically expressed proteins, have shown colocalisation of both KAP1 and repressed reporter target genes to domains of constitutive heterochromatin in the nucleus. However, we show here that although KAP1 does indeed become recruited to pericentric heterochromatin during differentiation of mouse embryonic stem (ES) cells, endogenous KRAB-ZFPs do not. Rather, KRAB-ZFPs and KAP1 relocalise to novel nucleoplasmic foci that we have termed KRAB- and KAP1-associated (KAKA) foci. HP1s can also concentrate in these foci and there is a close spatial relationship between KAKA nuclear foci and PML nuclear bodies. Finally, we reveal differential requirements for the recruitment of KAP1 to pericentric heterochromatin and KAKA foci, and suggest that KAKA foci may contain sumoylated KAP1 - the form of the protein that is active in transcriptional repression.

Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

DNA adduction by the potent carcinogen aflatoxin B1 : mechanistic studies

Aflatoxin B1, a potently carcinogenic fungal metabolite, is converted to the biologically active form by chemical oxidation using dimethyldioxirane and enzymatically by cytochrome P450 mixed-function oxidases. Both processes give rise to mixtures of the exo- and endo-8,9-epoxides. Methanolysis studies reveal exclusive trans opening of both epoxides under neutral conditions in CH3OH and CH3OH/H2O mixtures; an SN2 mechanism is postulated. Under acidic conditions, the exo isomer gives mixtures of trans and cis solvolysis products, suggesting that the reaction is, at least in part, SN1; the endo isomer gives only the trans product. The exo isomer reacts with DNA by attack of the nitrogen atom at the 7 position of guanine on C8 of the epoxide to give the trans adduct; the endo epoxide fails to form an adduct at this or any other site in DNA. The exo isomer is strongly mutagenic in a base-pair reversion assay employing Salmonella typhimurium; the endo isomer is essentially nonmutagenic. Aflatoxin B1 and its derivatives intercalate in DNA. These results are consistent with a mechanism in which intercalation of the exo epoxide optimally orients the epoxide for an SN2 reaction with guanine but intercalation of the endo isomer places the epoxide in an orientation which precludes reaction. Thus, while the exo epoxide is a potent mutagen, the endo epoxide fails to react with DNA.

Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection : a biomarker of oxidative DNA damage upon kidney transplantation

Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35 + 0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

Re-evaluation of the effect of smoking on the methylation of N-terminal valine in haemoglobin

In view of the established extrapulmonary cancer sites targeted by smoking a multiplicity of compounds, and mechanisms might be involved. It has been debated that smoking caused increased incidence of N-methylvaline at the N-terminus of haemoglobin. Because this could indicate a relevance of methylating nitrosamines in tobacco smoke, data are presented from an industrial cohort of 35 smokers and 21 non-smokers repeatedly monitored between 1994 and 1999. In general, N-methylvaline adduct levels in haemoglobin of smokers were approximately 50% higher than those of non-smokers. The smoking-induced methylation of haemoglobin is likely to be caused by dimethylnitrosamine (N-nitroso-dimethylamine), a major nitrosamine in side-stream tobacco smoke. The biomonitoring data emphasise the potential value of N-methylvaline as a smoking-related biomarker and call for intensified research on tobacco smoke compounds that lead to macromolecular methylation process.

DNA binding study of isophorone in rats and mice

Following isophorone exposure, in a 2-year study with F344 rats and B6C3F1 mice performed under the National Toxicology Program (NTP), an elevated incidence of tumors was observed in male rats (kidney tumors) and male mice (liver tumors). Female rats and mice showed no elevation of tumor rates by isophorone (NTP 1986).

Tumor-suppressor gene promoter hypermethylation in saliva of head and neck cancer patients

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80%(with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high. concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.

Epigenetics and migraine; complex mitochondrial interactions contributing to disease susceptibility

Migraine is a common neurological disorder classified by the World Health Organisation (WHO) as one of the top twenty most debilitating diseases in the developed world. Current therapies are only effective for a proportion of sufferers and new therapeutic targets are desperately needed to alleviate this burden. Recently the role of epigenetics in the development of many complex diseases including migraine has become an emerging topic. By understanding the importance of acetylation, methylation and other epigenetic modifications, it then follows that this modification process is a potential target to manipulate epigenetic status with the goal of treating disease. Bisulphite sequencing and methylated DNA immunoprecipitation have been used to demonstrate the presence of methylated cytosines in the human D-loop of mitochondrial DNA (mtDNA), proving that the mitochondrial genome is methylated. For the first time, it has been shown that there is a difference in mtDNA epigenetic status between healthy controls and those with disease, especially for neurodegenerative and age related conditions. Given co-morbidities with migraine and the suggestive link between mitochondrial dysfunction and the lowered threshold for triggering a migraine attack, mitochondrial methylation may be a new avenue to pursue. Creative thinking and new approaches are needed to solve complex problems and a systems biology approach, where multiple layers of information are integrated is becoming more important in complex disease modelling.