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Complexes incorporating a threading anthraquinone intercalator with pyrrole lexitropsin and platinum(II) moieties attached were developed with the goal of generating novel DNA binding modes, including the targeting of AT-rich regions in order to have high cytotoxicities. The binding of the complexes to DNA has been investigated and profiles surprisingly similar to that for cisplatin were observed; the profiles were different to those for a complex lacking the pyrrole lexitropsin component. The lack of selective binding to AT-rich regions suggests the platinum binding was dominating the sequence selectivity, and is consistent with the pyrrole lexitropsin slowing intercalation. The DNA unwinding profiles following platinum binding were evaluated by gel electrophoresis and suggested that intercalation and platinum binding were both occurring.

Netropsin is one of the first ligands to be discovered that selectively binds to the minor groove of DNA and is actively used as a scaffold for developing potential anticancer and antibiotic agents. The mechanism by which netropsin binds to hairpin DNA remains controversial with two competing mechanisms having been proposed. In one mechanism, netropsin binding induces a hairpin-to-duplex DNA transition. Alternatively, netropsin binds in two thermodynamically different modes at a single duplexed AATT site. Here, results from native mass spectrometry (MS) with nanoscale ion emitters indicate that netropsin can simultaneously and sequentially bind to both hairpin and duplex DNA. Duplex DNA was not detected using conventional MS with larger emitters because nanoscale emitters significantly reduce the extent of salt adduction to ligand-DNA complex ions, including in the presence of relatively high concentrations of nonvolatile salts. Based on native MS and polyacrylamide gel electrophoresis results, the abundances of hairpin and duplex DNA are unaffected by the addition of netropsin. By native MS, the binding affinities for five ligand-DNA and DNA-DNA interactions can be rapidly obtained simultaneously. This research indicates a "simultaneous binding mechanism" for the interactions of netropsin with DNA.

Purpose: The aim of this paper was to investigate the sequence preference of ionizing radiation (IR)-induced DNA damage as assessed by a linear amplification/polymerase stop (LA/PS) assay. The LA/PS assay is able to detect a wide range of IR-induced DNA lesions and this technique was utilized to quantitatively determine the preferential sites of gamma irradiation-induced DNA lesions in three different DNA sequences.Materials and methods: This analysis was performed on an automated DNA sequencer with capillary electrophoresis and laser-induced fluorescence detection.Results: The main outcome of this study was that G nucleotides were preferentially found at IR-induced polymerase stop sites. The individual nucleotides at the IR-induced DNA damage sites were analyzed and a consensus sequence of 5'-GG* (where * indicates the damaged nucleotide) was observed. In a separate method of analysis, the dinucleotides and trinucleotides at the IR-induced DNA damage sites were examined and 5'-GG* and 5'-G*G dinucleotides and 5'-GG*G trinucleotides were found to be the most prevalent. The use of the LA/PS assay permits a large number of IR-induced DNA lesions to be detected in the one procedure including: double- and single-strand breaks, apurinic/apyrimidinic sites and base damage.Conclusions: It was concluded that 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-G) and the degradation products of 8-oxoG were possibly the main lesions detected. To our knowledge, this is the first occasion that the DNA sequence preference of IR-induced DNA damage as detected by a LA/PS assay has been reported.

For ionising radiation (IR)-induced cellular toxicity, DNA cleavage is thought to be a crucial step. In this paper, the genome-wide DNA sequence preference of gamma radiation-induced cleavage was investigated in purified human DNA. We utilised Illumina short read technology and over 80 million double-strand breaks (DSBs) were analysed in this study. The frequency of occurrence of individual nucleotides at the 50,000 most frequently cleaved sites was calculated and C nucleotides were found to be most prevalent at the cleavage site, followed by G and T, with A being the least prevalent. 5'-C*C and 5'-CC* dinucleotides (where * is the cleavage site) were found to be the present at the highest frequency at the cleavage site; while it was 5'-CC*C for trinucleotides and 5'-GCC*C and 5'-CC*CC for tetranucleotides. The frequency of occurrence of individual nucleotides at the most frequently cleaved sites was determined and the nucleotides in the sequence 5'-GGC*MH (where M is A or C, H is any nucleotide except G) were found to occur most frequently for DNA that was treated with endonuclease IV (to remove blocking 3'-phosphoglycolate termini); and 5'-GSC*MH (where S is G or C) for non-endonuclease IV-treated DNA. It was concluded that GC-rich sequences were preferentially targeted for cleavage by gamma irradiation. This was the first occasion that an extensive examination of the genome-wide DNA sequence preference of IR-induced DSBs has been performed.

Background Cisplatin has been widely used for the treatment of cancer and its antitumour activity is attributed to its capacity to form DNA adducts, predominantly at guanine residues, which impede cellular processes such as DNA replication and transcription. However, there are associated toxicity and drug resistance issues which plague its use. This has prompted the development and screening of a range of chemotherapeutic drug analogues towards improved efficacy. The biological properties of three novel platinum-based compounds consisting of varying cis-configured ligand groups, as well as a commercially supplied compound, were characterised in this study to determine their potential as anticancer agents. Methods The linear amplification reaction was employed, in conjunction with capillary electrophoresis, to quantify the sequence specificity of DNA adducts induced by these compounds using a DNA template containing telomeric repeat sequences. Additionally, the DNA interstrand cross-linking and unwinding efficiency of these compounds were assessed through the application of denaturing and native agarose gel electrophoresis techniques, respectively. Their cytotoxicity was determined in HeLa cells using a colorimetric cell viability assay. Results All three novel platinum-based compounds were found to induce DNA adduct formation at the tandem telomeric repeat sequences. The sequence specificity profile at these sites was characterised and these were distinct from that of cisplatin. Two of these compounds with the enantiomeric 1,2-diaminocyclopentane ligand (SS and RR-DACP) were found to induce a greater degree of DNA unwinding than cisplatin, but exhibited marginally lower DNA cross-linking efficiencies. Furthermore, the RR-isomer was more cytotoxic in HeLa cells than cisplatin. Conclusions The biological characteristics of these compounds were assessed relative to cisplatin, and a variation in the sequence specificity and a greater capacity to induce DNA unwinding was observed. These compounds warrant further investigations towards developing more efficient chemotherapeutic drugs.

Bleomycin (BLM) is a cancer chemotherapeutic agent that cleaves cellular DNA at specific sequences. Using next-generation Illumina sequencing, the genome-wide sequence specificity of DNA cleavage by two BLM analogues, 6′-deoxy-BLM Z and zorbamycin (ZBM), was determined in human HeLa cells and compared with BLM. Over 200 million double-strand breaks were examined for each sample, and the 50,000 highest intensity cleavage sites were analysed. It was found that the DNA sequence specificity of the BLM analogues in human cells was different to BLM, especially at the cleavage site (position “0”) and the “+1” position. In human cells, the 6′-deoxy-BLM Z had a preference for 5′-GTGY*MC (where * is the cleavage site, Y is C or T, M is A or C); it was 5′-GTGY*MCA for ZBM; and 5′-GTGT*AC for BLM. With cellular DNA, the highest ranked tetranucleotides were 5′-TGC*C and 5′-TGT*A for 6′-deoxy-BLM Z; 5′-TGC*C, 5′-TGT*A and 5′-TGC*A for ZBM; and 5′-TGT*A for BLM. In purified human genomic DNA, the DNA sequence preference was 5′-TGT*A for 6′-deoxy-BLM, 5′-RTGY*AYR (where R is G or A) for ZBM, and 5′-TGT*A for BLM. Thus, the sequence specificity of the BLM analogue, 6′-deoxy-BLM Z, was similar to BLM in purified human DNA, while ZBM was different.

In this work, we examined the DNA sequence preference of gamma-radiation-induced DNA damage in purified DNA sequences after heat treatment. DNA was fluorescently end-labeled and gamma-radiation-induced DNA cleavage was examined using capillary electrophoresis with laser-induced fluorescence detection. Our findings provide evidence that gamma-radiation-induced DNA damage to end-labeled DNA is nonrandom and has a sequence preference. The degree of cleavage was quantified at each nucleotide, and we observed that preferential cleavage occurred at C nucleotides with lesser cleavage at G nucleotides, while being very low at T nucleotides. The differences in percentage cleavage at individual nucleotides ranged up to sixfold. The DNA sequences surrounding the most intense radiation-induced DNA cleavage sites were examined and a consensus sequence 5'-AGGC*C (where C* is the cleavage site) was found. The highest intensity gamma-radiation-induced DNA cleavage sites were found at the dinucleotides, 5'-GG*, 5'-GC*, 5'-C*C and 5'-G*G and at the trinucleotides, 5'-GG*C, 5'-TC*A, 5'-GG*G and 5'-GC*C. These findings have implications for our understanding of ionizing radiation-induced DNA damage.

The presence of adducts on the DNA double-helix can have major consequences for the efficient functioning of DNA repair enzymes. E. coli RecBCD (exonuclease V) is involved in recombinational repair of double-strand breaks that are caused by defective DNA replication, DNA damaging agents and other factors. The holoenzyme possesses a bipolar helicase activity which helps unwind DNA from both 3′- and 5′-directions and is coupled with a potent exonuclease activity that is also capable of digesting DNA from both 3′- and 5′-ends. In this study, DNA sequences were damaged with cisplatin or UV followed by RecBCD treatment. DNA damaging agents such as cisplatin and UV induce the formation of intrastrand adducts in the DNA template. It was demonstrated that RecBCD degradation was inhibited by either cisplatin-damaged or UV-damaged DNA sequences. This is the first occasion that RecBCD has been demonstrated to be inhibited by DNA adducts induced by cisplatin or UV. In addition, we quantified the amounts of DNA remaining after RecBCD treatment and observed that the level of inhibition was concentration and dose dependent. A DNA-targeted 9-aminoacridinecarboxamide cisplatin analogue was also found to inhibit RecBCD activity.

Ultraviolet light (UV) is one of the most common DNA damaging agents in the human environment. This paper examined the influence of DNA methylation on the level of UVB- and UVC-induced DNA damage. A purified DNA sequence containing CpG dinucleotides was methylated with a CpG methylase. We employed the linear amplification technique and the end-labelling approach followed by capillary electrophoresis with laser-induced fluorescence to investigate the sequence specificity of UV-induced DNA damage. The linear amplification technique mainly detects cyclobutane pyrimidine dimer (CPD) adducts, while the end-labelling approach mainly detects 6–4 photoproduct (6–4PP) lesions. The levels of CPD and 6–4PP adducts detected in methylated/unmethylated labelled sequences were analysed. The comparison showed that 5-methyl-cytosine significantly reduced the level of both CPD and 6–4PP adducts after UVB (308 nm) and UVC (254 nm) irradiation compared with the non-methylated counterpart.

Cisplatin analogues with an attached DNA-binding moiety represent a potentially effective class of DNA-damaging anti-tumour agents because they possess higher affinities for DNA and different DNA damage profiles compared with cisplatin. In this study, the interaction of four 9-aminoacridine carboxamide Pt complexes with purified DNA was investigated: firstly, using a fluorescent intercalator displacement (FID) assay with ethidium bromide; and secondly, with a DNA unwinding assay. The relative capacity of these compounds to perturb the fluorescence induced by DNA-bound ethidium bromide at clinically relevant drug concentrations was assessed over a 24-h period using an FID assay. All analogues were found to reduce the level of ethidium bromide-induced fluorescence in a concentration-dependent manner from the earliest time point of 10 min onwards. Cisplatin, however, showed a markedly slower reduction in ethidium bromide-induced fluorescence from 2 h onwards, producing a similar level of fluorescence reduction as that produced by the analogues from 6 h onwards. These results suggest that the altered DNA-binding modes of the DNA-targeted analogues confer a more efficient mechanism for DNA binding compared with cisplatin. Relative DNA binding coefficients were also determined for each of the compounds studied. With the DNA unwinding assay, an unwinding angle can be calculated from the coalescence point of plasmids in an agarose gel. It was found that all 9-aminoacridine carboxamide analogues had a greater unwinding angle compared with cisplatin. The knowledge obtained from these two assays has helped to further characterise the cisplatin analogues and could facilitate the development of more effective anti-tumour agents.

Cisplatin is widely used as a cancer chemotherapeutic agent and this review covers the mechanism of action of cisplatin, cellular resistance to cisplatin, the genomic location of cisplatin adducts and the properties of DNA-targeted Pt complexes. A particular focus of this review is the interaction of Pt compounds with DNA. The technology involved in determining Pt-drug/DNA interactions has advanced and permits clearer views of this process. In particular, molecular biological techniques permit a more accurate and precise determination of the sequence specific preference of Pt adduct formation. Prospects for the sequence specific genome-wide determination of Pt adduct formation using next-generation sequencing are also discussed. Cisplatin analogues that are targeted to DNA via an attached DNA-affinic moiety are potentially beneficial anti-tumour agents. In particular the 9-aminoacridine Pt complexes possess a number of important characteristics, including activity against cisplatin-resistant cells. Their ability to circumvent resistance due to increased DNA repair may allow these DNA-targeted analogues to avoid many of the drawbacks associated with current clinical oncology treatment. This ability is thought to be due to their altered DNA sequence specificity, compared with cisplatin, where Pt adduct formation for the 9- aminoacridine Pt complexes was shifted away from consecutive guanines towards 5'-CG and 5'-GA dinucleotide sequences. Evidence for this evasion of repair processes and avoidance of cellular cisplatin resistance was found for 9-aminoacridine Pt complexes in studies with cisplatin resistant cells. The prospects for clinical use of these DNA-targeted anti-tumour agents were evaluated.

Ionising radiation (IR) is known to induce a wide variety of lesions in DNA. In this review, we compared three different techniques that examined the DNA sequence preference of IR-induced DNA damage at nucleotide resolution. These three techniques were: the linear amplification/polymerase stop assay, the end-labelling procedure, and Illumina next-generation genome-wide sequencing. The DNA sequence preference of IR-induced DNA damage was compared in purified DNA sequences including human genomic DNA. It was found that the DNA sequence preference of IR-induced DNA damage identified by the end-labelling procedure (that mainly detected single-strand breaks) and Illumina next-generation genome-wide sequencing (that mainly detected double-strand breaks) was at C nucleotides, while the linear amplification/polymerase stop assay (that mainly detected base damage) was at G nucleotides. A consensus sequence at the IR-induced DNA damage was found to be 5&prime, AGGC*C for the end-labelling technique, 5&prime, GGC*MH (where * is the cleavage site, M is A or C, H is any nucleotide except G) for the genome-wide technique, and 5&prime, GG* for the linear amplification/polymerase stop procedure. These three different approaches are important because they provide a deeper insight into the mechanism of action of IR-induced DNA damage.

Bleomycin (BLM) is used clinically in combination with a number of other agents for the treatment of several types of tumours. Members of the BLM family of drugs include zorbamycin (ZBM), phleomycin D1, BLM A2 and BLM B2. By manipulating the BLM biosynthetic machinery, we have produced two new BLM analogues, BLM Z and 6′-deoxy-BLM Z, with the latter exhibiting significantly improved DNA cleavage activity. Here we determined the DNA sequence specificity of BLM Z, 6′-deoxy-BLM Z and ZBM, in comparison with BLM, with high precision using purified plasmid DNA and our recently developed technique. It was found that ZBM had a different DNA sequence specificity compared with BLM and the BLM analogues. While BLM and the BLM analogues showed a similar DNA sequence specificity, with TGTA sequences as the main site of cleavage, ZBM exhibited a distinct DNA sequence specificity, with both TGTA and TGTG as the predominant cleavage sites. These differences in DNA sequence specificity are discussed in relation to the structures of ZBM, BLM and the BLM analogues. Our findings support the strategy of manipulating the BLM biosynthetic machinery for the production of novel BLM analogues, difficult to prepare by total synthesis; some of which could have beneficial cancer chemotherapeutic properties.

This study investigated the effect of CpG methylation on the DNA binding of cisplatin analogues with an attached aminoacridine intercalator. DNA-targeted 9-aminoacridine carboxamide Pt complexes are known to bind at 5'-CpG sequences. Their binding to methylated and non-methylated 5'-CpG sequences was determined and compared with cisplatin. The damage profiles of each platinum compound were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. Methylation at 5'-CpG was shown to significantly increase the binding intensity for the 9-aminoacridine carboxamide compounds, whereas no significant increase was found for cisplatin. 5'-CpG methylation had the largest effect on the 9-ethanolamine-acridine carboxamide Pt complex, followed by the 9-aminoacridine carboxamide Pt complex and the 7-fluoro complex. The methylation state of a cell's genome is important in maintaining normal gene expression, and is often aberrantly altered in cancer cells. An analogue of cisplatin which differentially targets methylated DNA may be able to improve its therapeutic activity, or alter its range of targets and evade the chemoresistance which hampers cisplatin efficacy in clinical use.

The cancer chemotherapeutic agent, bleomycin, cleaves DNA at specific sites. For the first time, the genome-wide DNA sequence specificity of bleomycin breakage was determined in human cells. Utilising Illumina next-generation DNA sequencing techniques, over 200 million bleomycin cleavage sites were examined to elucidate the bleomycin genome-wide DNA selectivity. The genome-wide bleomycin cleavage data were analysed by four different methods to determine the cellular DNA sequence specificity of bleomycin strand breakage. For the most highly cleaved DNA sequences, the preferred site of bleomycin breakage was at 5'-GT* dinucleotide sequences (where the asterisk indicates the bleomycin cleavage site), with lesser cleavage at 5'-GC* dinucleotides. This investigation also determined longer bleomycin cleavage sequences, with preferred cleavage at 5'-GT*A and 5'- TGT* trinucleotide sequences, and 5'-TGT*A tetranucleotides. For cellular DNA, the hexanucleotide DNA sequence 5'-RTGT*AY (where R is a purine and Y is a pyrimidine) was the most highly cleaved DNA sequence. It was striking that alternating purine-pyrimidine sequences were highly cleaved by bleomycin. The highest intensity cleavage sites in cellular and purified DNA were very similar although there were some minor differences. Statistical nucleotide frequency analysis indicated a G nucleotide was present at the -3 position (relative to the cleavage site) in cellular DNA but was absent in purified DNA.

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, was determined with high precision in purified plasmid DNA using an improved technique. This improved technique involved the labelling of the 5'- and 3'-ends of DNA with different fluorescent tags, followed by simultaneous cleavage by bleomycin and capillary electrophoresis with laser-induced fluorescence. This permitted the determination of bleomycin cleavage specificity with high accuracy since end-label bias was greatly reduced. Bleomycin produces single- and double-strand breaks, abasic sites and other base damage in DNA. This high-precision method was utilised to elucidate, for the first time, the DNA sequence specificity of bleomycin-induced DNA damage at abasic sites. This was accomplished using endonuclease IV that cleaves DNA at abasic sites after bleomycin damage. It was found that bleomycin-induced abasic sites formed at 5'-GC and 5'-GT sites while bleomycin-induced phosphodiester strand breaks formed mainly at 5'-GT dinucleotides. Since bleomycin-induced abasic sites are produced in the absence of molecular oxygen, this difference in DNA sequence specificity could be important in hypoxic tumour cells.

Objective: An important but neglected consequence of the AIDS pandemic that continues across sub-Saharan Africa is the phenomenon of child-headed households (CHH). This study aims to describe the challenges to health and well-being for young people living in child-headed households.Methods: A mixed-methods research approach linked common themes using qualitative and quantitative instruments to provide a broad picture of the location and challenges of CHH in Kabira, Kyotera and Kamuganja in the Rakai District of southern Uganda. Local knowledge was used to locate CHH.Results: 163 children living in 40 CHH were traced: 42·5% of the household heads were double orphans caring for younger siblings, and 43% were also caring for chronically ill or disabled grandparents who were economically unproductive and largely dependent on the eldest child for survival. It was found that those heading households were more likely not to attend school than children living at home with a parent. Their immediate needs ranged from food and shelter to health-care and education. Fear was a major theme: 38% of those interviewed reported fear of ‘violence’. Children as young as 13 were responsible for navigating through complex decision-making processes from everyday basic necessities to decisions on the health care of younger siblings and grandparents.Conclusion: Children and young people living in CHH are a largely invisible and highly vulnerable population. Clear, officially accepted definitions of CHH are a first step in recognising this vulnerable group for whom safeguards will be necessary as social work develops in lower- and middle-income countries (LMICs). The precise numbers of CHH are unknown and further examination of this undocumented group is needed.

Irish intercultural cinema looks at the development of a cinematic genre which focuses on issues of Irish migrancy but is produced outside of Ireland. This paper has as its focus the cultural landscape of Irish-Australia. The essay uses methodologies of ethnographic and documentary theory plus textual analysis of film and written texts to establish a throughline of Irish intercultural film. The essay begins by contextualising the place of the Irish diaspora within the creation of Irish identity globally. The discussion around migrancy is widened to consider the place of memory and intergenerational tensions within not just the Irish migrant population, but also within the diverse cultures which comprise the contemporary Australian landscape. The historical development of intercultural cinema is then explored internationally within a context of colonial, gender and class struggles in the 1970s and1980s. The term intercultural cinema has its origins in the Third Cinema of Argentinians Solanas and Getino in the 1970s and covers those films which deal with issues involving two countries or cultures. The term was refined by Laura Marks in 2000 and further developed by Hamid Naficy in 2001 in his discussion of accented cinema which narrows its definition to include the politics of production. The paper then traces the development of Irish intercultural cinema from its beginnings in England in the 1970s with Thaddeus O'Sullivan through to Nicola Bruce and others including Enda Murray in the present day. The essay concludes by bringing these various strands together to see where intercultural film might have a place in today's globalised cultural landscape. Common traits within intercultural film such as the notion of place, autobiographical film and personal identity are explored using examples of intercultural filmmaking from around the globe. These commonalities point to a way forward for the future of a sustainable multicultural film culture.

The cancer chemotherapeutic drug, bleomycin, is clinically used to treat several neoplasms including testicular and ovarian cancers. Bleomycin is a metallo-glycopeptide antibiotic that requires a transition metal ion, usually Fe(II), for activity. In this review, the properties of bleomycin are examined, especially the interaction of bleomycin with DNA. A Fe(II)-bleomycin complex is capable of DNA cleavage and this process is thought to be the major determinant for the cytotoxicity of bleomycin. The DNA sequence specificity of bleomycin cleavage is found to at 5′-GT* and 5′-GC* dinucleotides (where * indicates the cleaved nucleotide). Using next-generation DNA sequencing, over 200 million double-strand breaks were analysed, and an expanded bleomycin sequence specificity was found to be 5′-RTGT*AY (where R is G or A and Y is T or C) in cellular DNA and 5′-TGT*AT in purified DNA. The different environment of cellular DNA compared to purified DNA was proposed to be responsible for the difference. A number of bleomycin analogues have been examined and their interaction with DNA is also discussed. In particular, the production of bleomycin analogues via genetic manipulation of the modular non-ribosomal peptide synthetases and polyketide synthases in the bleomycin gene cluster is reviewed. The prospects for the synthesis of bleomycin analogues with increased effectiveness as cancer chemotherapeutic agents is also explored.

The sequence specificity of UV-induced DNA damage was investigated in a specifically designed DNA plasmid using two procedures: end-labelling and linear amplification. Absorption of UV photons by DNA leads to dimerisation of pyrimidine bases and produces two major photoproducts, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). A previous study had determined that two hexanucleotide sequences, 5'-GCTC*AC and 5'-TATT*AA, were high intensity UV-induced DNA damage sites. The UV clone plasmid was constructed by systematically altering each nucleotide of these two hexanucleotide sequences. One of the main goals of this study was to determine the influence of single nucleotide alterations on the intensity of UV-induced DNA damage. The sequence 5'-GCTC*AC was designed to examine the sequence specificity of 6-4PPs and the highest intensity 6-4PP damage sites were found at 5'-GTTC*CC nucleotides. The sequence 5'-TATT*AA was devised to investigate the sequence specificity of CPDs and the highest intensity CPD damage sites were found at 5'-TTTT*CG nucleotides. It was proposed that the tetranucleotide DNA sequence, 5'-YTC*Y (where Y is T or C), was the consensus sequence for the highest intensity UV-induced 6-4PP adduct sites; while it was 5'-YTT*C for the highest intensity UV-induced CPD damage sites. These consensus tetranucleotides are composed entirely of consecutive pyrimidines and must have a DNA conformation that is highly productive for the absorption of UV photons.

Fluorinated nucleoside analogues are a major class of cancer chemotherapy agents, and include the drugs 5-fluorouracil (5FU) and 5-fluoro-2'-deoxyuridine (FdUrd). The aim of this study was to examine the cellular toxicity of two novel fluorinated pyrimidine L-nucleosides that are enantiomers of D-nucleosides and may be able to increase selectivity for cancer cells as a result of their unnatural L-configuration. Two fluorinated pyrimidine L-nucleosides were examined in this study, L110 ([β-L, β-D]-5-fluoro-2'-deoxyuridine) and L117 (β-L-deoxyuridine:β-D-5'-fluoro-2'-deoxyuridine). The cytotoxicity of these L-nucleoside was determined in primary mouse fibroblasts and was compared with 5FU and FdUrd. In addition, the influence of p53 status on cytotoxicity was investigated. These cytotoxicity assays were performed on a matched set of primary mouse fibroblasts that were either wild type or null for the p53 tumour suppressor gene. It was found that cells lacking functional p53 were over 7500 times more sensitive to the drugs L110, L117 and FdUrd than cells containing wild type p53.

Tandem repeats of short DNA sequences are commonly found in human DNA. These simple sequence repeats or microsatellites are highly polymorphic in the human genome. Since the anti-tumour agent cisplatin preferentially forms DNA adducts at runs of consecutive guanine nucleotides (poly(G)), the position and frequency of occurrence of poly(G) sequences in the updated human genome was investigated. There are more runs of consecutive guanines than would be expected by random chance. This especially true for poly(G) sequences longer than approximately n=9. A plot of poly(G) length against log(observed/expected) frequency produced a straight line for n>9. A similar observation was also found for poly(A) DNA sequence repeats. This data implied that the increase in observed/expected frequency is directly related to length of DNA repeat. It was proposed that long runs of consecutive guanine nucleotides could be a sensitive sensor of cellular DNA damage since a number of DNA damaging agents cause lesions at poly(G) sequences.

The enzyme T4 endonuclease VII is a resolvase that acts on branched DNA intermediates during genetic recombination, by cleaving DNA with staggered cuts approximately 3–6 bp apart. In this paper, we investigated the sequence preference of this cleavage reaction utilising two different DNA sequences. For the first time, the DNA sequence preference of T4 endonuclease VII cleavage sites has been examined without the presence of a known DNA substrate to mask any inherent nucleotide preference. The use of the ABI3730 platform enables the cleavage site to be determined at nucleotide resolution. We found that T4 endonuclease VII cleaves DNA with a sequence preference. We calculated the frequency of nucleotides surrounding the cleavage sites and found that following nucleotides had the highest incidence: AWTAN*STC, where N* indicates the cleavage site between positions 0 and 1, N is any base, W is A or T, and S is G or C. An A at position −1 and T at position +2 were the most predominant nucleotides at the cleavage site. Using a Sequence Logo method, the sequence TATTAN*CT was derived at the cleavage site. Note that A and T nucleotides were highly preferred 5′ to the cleavage sites in both methods of analysis. It was proposed that the enzyme recognises the narrower minor groove of these consecutive AT base pairs and cleaves DNA 3′ to this feature.